Antisense antibacterial method and composition

ABSTRACT

The invention relates to compositions comprising oligomers antisense to bacterial 16S or 23S rRNA and capable of selectively modulating the biological activity thereof, and methods for their use. More particularly, the invention relates to antisense oligomers directed to 16S or 23S rRNA found in one or more particular bacteria, or generally conserved among bacteria in general, and to pharmaceutical compositions and methods of treatment comprising the same.

This application claims benefit of U.S. Provisional Application No.60/168,150, filed Nov. 29, 1999, which is incorporated in its entiretyherein by reference.

FIELD OF THE INVENTION

The present invention relates to oligonucleotide compositions antisenseto bacterial 16S and 23S rRNA and methods for use of such compositionsin the treatment of bacterial infection in a mammal.

REFERENCES

Agrawal, S. et al., Proc. Natl. Acad. Sci. USA 87(4):1401-5 (1990).

Ardhammar, M. et al., J. Biomolecular Structure & Dynamics 17(1):3340(August 1999).

Attia, S. A. et al., Antisense & Nucleic Acid Drug Dev. 8 (3):207-14(1998).

Bennett, M. R. et al., Circulation 92(7):1981-1993 (1995).

Bonham, M. A. et al., Nucleic Acids Res. 23(7):1197-1203 (1995).

Boudvillain, M. et al., Biochemistry 36(10):2925-31 (1997).

Cross, C. W. et al., Biochemistry 36(14):4096-107 (Apr. 8 1997).

Dagle, J. M. et al., Nucleic Acids Research 28(10):2153-7 (May 15,2000).

Ding, D. et al., Nucleic Acids Research 24(2):354-60 (Jan. 15, 1996).

Egholm, M. et al., Nature 365(6446):566-8 (Oct. 7, 1993).

Felgner et al., Proc. Nat. Acad. Sci. USA 84:7413 (1987).

Gait, M. J.; Jones, A. S. and Walker, R. T., J. Chem. Soc. Perkin I,1684-86 (1974).

Gee, J. E. et al., Antisense & Nucleic Acid Drug Dev. 8:103-111 (1998).

Good, L. and Nielsen, P. E., Proc. Nat. Acad. Sci. USA 95:2073-2076(1998).

Huie, E. M. et al., J. Org. Chem. 57:4569 (1992).

Jones, A. S., MacCross, M. and Walker, R. T., Biochem. Biophys. Acta365:365-377 (1973).

Lesnikowski, Z. J. et al., Nucleic Acids Research 18(8):2109-15 (Apr. 251990).

Matteucci, M., Tetrahedron Lett. 31:2385-88 (1990).

McElroy, E. B. et al., Bioorg. Med. Chem. Lett. 4:1071 (1994).

Mertes, M. P. and Coates, E. A., J. Med. Chem. 12:154-157 (1969).

Miller, P. S. et al., in: Antisense Research Applications, Crooke, S. T.and Lebleu, B., Eds., CRC Press, Boca Raton, Fla., p. 189. (1993).

Olgive, K. K. and Cormier, J. F., Tetrahedron Lett 26:4159-4162 (1986).

Rahman, M. A. et al., Antisense Res Dev 1(4):319-27 (1991).

Roughton, A. L. et al., J. Am. Chem. Soc. 117:7249 (1995).

Stein, D. et al., Antisense & Nucleic Acid Drug Dev. 7(3):151-7 (June1997); see also

Summerton, J. et al., Antisense & Nucleic Acid Drug Dev. 7(2):63-70(April 1997).

Toulme, J. J. et al., Biochimie 78(7):663-73 (1996).

Vasseur, J. J. et al., J. Am. Chem. Soc. 114:4006 (1992).

BACKGROUND OF THE INVENTION

Currently, there are several types of antibiotics in use againstbacterial pathogens, with a variety of anti-bacterial mechanisms.Beta-lactam antibiotics, such as penicillin and cephalosporin, act toinhibit the final step in peptidoglycan synthesis. Glycopeptideantibiotics, including vancomycin and teichoplanin, inhibit bothtransglycosylation and transpeptidation of muramyl-pentapeptide, againinterfering with peptidoglycan synthesis. Other well-known antibioticsinclude the quinolones, which inhibit bacterial DNA replication,inhibitors of bacterial RNA polymerase, such as rifampin, and inhibitorsof enzymes in the pathway for production of tetrahydrofolate, includingthe sulfonamides.

Some classes of antibiotics act at the level of protein synthesis.Notable among these are the aminoglycosides, such as kanamycin andgentamycin. These compounds target the bacterial 30S ribosome subunit,preventing the association with the 50S subunit to form functionalribosomes. Tetracyclines, another important class of antibiotics, alsotarget the 30S ribosome subunit, acting by preventing alignment ofaminoacylated tRNA's with the corresponding mRNA codon. Macrolides andlincosamides, another class of antibiotics, inhibit bacterial synthesisby binding to the 50S ribosome subunit, and inhibiting peptideelongation or preventing ribosome translocation.

Despite impressive successes in controlling or eliminating bacterialinfections by antibiotics, the widespread use of antibiotics both inhuman medicine and as a feed supplement in poultry and livestockproduction has led to drug resistance in many pathogenic bacteria.Antibiotic resistance mechanisms can take a variety of forms. One of themajor mechanisms of resistance to beta lactams, particularly inGram-negative bacteria, is the enzyme beta-lactamase, which renders theantibiotic inactive. Likewise, resistance to aminoglycosides ofteninvolves an enzyme capable of inactivating the antibiotic, in this caseby adding a phosphoryl, adenyl, or acetyl group. Active efflux ofantibiotics is another way that many bacteria develop resistance. Genesencoding efflux proteins, such as the tetA, tetG, tetL, and tetK genesfor tetracycline efflux, have been identified. A bacterial target maydevelop resistance by altering the target of the drug. For example, theso-called penicillin binding proteins (PBPs) in many beta-lactamresistant bacteria are altered to inhibit the critical antibioticbinding to the target protein. Resistance to tetracycline may involve,in addition to enhanced efflux, the appearance of cytoplasmic proteinscapable of competing with ribosomes for binding to the antibiotic. Wherethe antibiotic acts by inhibiting a bacterial enzyme, such as forsulfonamides, point mutations in the target enzyme may conferresistance.

The appearance of antibiotic resistance in many pathogenic bacteria, inmany cases involving multi-drug resistance, has raised the specter of apre-antibiotic era in which many bacterial pathogens are simplyuntreatable by medical intervention. There are two main factors thatcould contribute to this scenario. The first is the rapid spread ofresistance and multi-resistance genes across bacterial strains, species,and genera by conjugative elements, the most important of which areself-transmissible plasmids. The second factor is a lack of currentresearch efforts to find new types of antibiotics, due in part to theperceived investment in time and money needed to find new antibioticagents and bring them through clinical trials, a process that mayrequire a 20-year research effort in some cases.

In addressing the second of these factors, some drug-discoveryapproaches that may accelerate the search for new antibiotics have beenproposed. For example, efforts to screen for and identify new antibioticcompounds by high-throughput screening have been reported, but to dateno important lead compounds have been discovered by this route.

Several approaches that involve antisense agents designed to block theexpression of bacterial resistance genes or to target cellular RNAtargets, such as the rRNA in the 30S ribosomal subunit, have beenproposed (Good et al., 1998; Rahman et al., 1991). In general, theseapproaches have been marginally successful, presumably because of pooruptake of the antisense agent (e.g., Summerton et al., 1997), or therequirement that the treated cells show high permeability forantibiotics (Good et al., 1998).

There is thus a growing need for new antibiotics that (i) are notsubject to the principal types of antibiotic resistance currentlyhampering antibiotic treatment of bacteria, (ii) can be developedrapidly and with some reasonable degree of predictability as totarget-bacteria specificity, (iii) can also be designed forbroad-spectrum activity, (iv) are effective at low doses, meaning, inpart, that they are efficiently taken up by wild-type bacteria or evenbacteria that have reduced permeability for antibiotics, and (v) showfew side effects.

SUMMARY OF THE INVENTION

In one aspect, the invention provides an antibacterial compound,consisting of a substantially uncharged antisense oligomer containingfrom 8 to 40 nucleotide subunits, including a targeting nucleic acidsequence at least 10 nucleotides in length which is complementary to abacterial 16S or 23S rRNA nucleic acid sequence. Each of the subunitscomprises a 5- or 6-membered ring supporting a base-pairing moietyeffective to bind by Watson-Crick base pairing to a respectivenucleotide base in the bacterial nucleic acid sequence. Adjacentsubunits are joined by uncharged linkages selected from the groupconsisting of: uncharged phosphoramidate, phosphorodiamidate, carbonate,carbamate, amide, phosphotriester, alkyl phosphonate, siloxane, sulfone,sulfonamide, sulfamate, thioformacetyl, andmethylene-N-methylhydroxylamino, or by charged linkages selected fromthe group consisting of phosphate, charged phosphoramidate andphosphorothioate. The ratio of uncharged linkages to charged linkages inthe oligomer is at least 4:1, preferably at least 5:1, and morepreferably at least 8:1. In one embodiment, the oligomer is fullyuncharged.

Preferably, the oligomer is able to hybridize with the bacterialsequence at a Tm substantially greater than the Tm of a duplex composedof a corresponding DNA and the same bacterial sequence. Alternatively,the oligomer is able to hybridize with the bacterial sequence at a T_(m)substantially greater than 37° C., preferably greater than 50° C., andmore preferably in the range of 60-80° C.

In one embodiment, the oligomer is a morpholino oligomer. The unchargedlinkages, and, in one embodiment, all of the linkages, in such anoligomer are preferably selected from the group consisting of thestructures presented in FIGS. 2A through 2D. Particularly preferred arephosphorodiamidate-linked oligomers, as represented at FIG. 2B, whereX═NR₂, R being hydrogen or methyl, Y═O, and Z═O.

The length of the oligomer is preferably 12 to 25 subunits. In oneembodiment, the oligomer is a phosphorodiamidate-linked morpholinooligomer having a length of 15 to 20 subunits, and more preferably 17-18subunits.

In selected embodiments, the targeting sequence is a broad spectrumsequence selected from the group consisting of SEQ ID NOS:15, 16, and21-25. In other embodiments, the targeting sequence is complementary toa Gram-positive bacterial 16S rRNA consensus sequence, e.g., SEQ IDNOS:27-28, or is complementary to a Gram-negative bacterial 16S rRNAconsensus sequence, e.g. SEQ ID NOS:29-30.

Other targeting sequences can be used for treatment of an infectionproduced by various organisms, for example:

(a) E. coli, where the sequence is selected from the group consisting ofSEQ ID NO:32 and SEQ ID NO:35;

(b) Salmonella thyphimurium, where the sequence is selected from thegroup consisting of SEQ ID NO:18 and SEQ ID NO:36;

(c) Pseudomonas aeruginosa, where the sequence is selected from thegroup consisting of SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ IDNO:43;

(d) Vibrio cholera, where the sequence is selected from the groupconsisting of SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47;

(e) Neisseria gonorrhoea, where the sequence is selected from the groupconsisting of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51;

(f) Staphylococcus aureus, where the sequence is selected from the groupconsisting of SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55;

(g) Mycobacterium tuberculosis, where the sequence is selected from thegroup consisting of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ IDNO:59;

(h) Helicobacter pylori, where the sequence is selected from the groupconsisting of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63;

(i) Streptococcus pneumoniae, where the sequence is selected from thegroup consisting of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 and SEQ IDNO:67;

(j) Treponema palladium, where the sequence is selected from the groupconsisting of SEQ ID NO:69, SEQ ID NO:70 and SEQ ID NO:71;

(k) Chlamydia trachomatis, where the sequence is selected from the groupconsisting of SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75;

(l) Bartonella henselae, where the sequence is selected from the groupconsisting of SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79;

(m) Hemophilis influenza, where the sequence is selected from the groupconsisting of SEQ ID NO:81, SEQ ID NO:82 and SEQ ID NO:83;

(n) Shigella dysenterae, where the sequence is presented as SEQ IDNO:88; or

(o) Enterococcus faecium, where the sequence is presented as SEQ IDNO:92.

In other embodiments, the targeting sequence is an antisense oligomersequence selected from one of the following groups, for use in treatmentof an infection produced by:

(a) E. coli, Salmonella thyphimurium and Shigella dysenterae, where thesequence is selected from the group consisting of SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:39 and SEQ IDNO:86 and SEQ ID NO:87;

(b) E. coli, Salmonella thyphimurium and Hemophilis influenza, where thesequence is presented as SEQ ID NO:31;

(c) E. coli and Shigella dysenterae, where the sequence is presented asSEQ ID NO:17;

(d) E. coli, Salmonella thyphimurium, Shigella dysenterae, Hemophilisinfluenza and Vibrio cholera, where the sequence is presented as SEQ IDNO:44;

(e) Staphylococcus aureus and Bartonella henselae, where the sequence ispresented as SEQ ID NO:52;

(f) Salmonella thyphimurium, Hemophilis influenza and Treponemapalladium, where the sequence is presented as SEQ ID NO:68; or

(g) E. coli, Salmonella thyphimurium, Shigella dysenterae, Hemophilisinfluenza and Neisseria gonorrhoea, where the sequence is presented asSEQ ID NO:84.

In a related aspect, the invention provides a method of treating abacterial infection in a human or mammalian animal subject, byadministering to the subject, in a pharmaceutically effective amount, asubstantially uncharged antisense oligomer as described above. Variousselected embodiments of the oligomer and the target sequence are asdescribed above. Preferably, the antisense oligomer is administered inan amount and manner effective to result in a peak blood concentrationof at least 200-400 nM antisense oligomer. The method can be used, forexample, for treating bacterial infections of the skin, whereinadministration is by a topical route, or for use in treating a bacterialrespiratory infection, wherein administration is by inhalation.

In a further related aspect, the invention provides a livestock andpoultry food composition containing a food grain supplemented with asubtherapeutic amount of an antibacterial compound, said compoundconsisting of a substantially uncharged antisense oligomer as describedabove.

Also contemplated is, in a method of feeding livestock and poultry witha food grain supplemented with subtherapeutic levels of an antibiotic,an improvement in which the food grain is supplemented with asubtherapeutic amount of an antibacterial compound of the type describedabove.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying figures and examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows several preferred morpholino-type subunits having 5-atom(A), six-atom (B) and seven-atom (C-D) linking groups suitable forforming polymers;

FIGS. 2A-D show the repeating subunit segment of exemplary morpholinooligonucleotides, designated A through D, constructed using subunitsA-D, respectively, of FIG. 1.

FIGS. 3A-3G show examples of uncharged linkage types in oligonucleotideanalogs;

FIG. 4 depicts the results of a study on the effect of aphosphorodiamidate morpholino antisense oligomer (PMO) designated VRE-2(SEQ ID NO:92) (see Table 10), targeted against an Enterococcus faecium16S rRNA sequence, alone or in combination with 50 μM of an oligomerantisense to c-myc (SEQ ID NO:139), on bacterial colony formation in E.coli, presented as percent viability;

FIG. 5 depicts the results of a study on the effect of variousconcentrations of a PMO having SEQ ID NO:15 (broad spectrum; see Table2A), targeted against a bacterial 16S rRNA consensus sequence, on thebacterial colony formation in E. coli, presented as percent inhibitionof colony formation;

FIG. 6 depicts the results of a study wherein PMO oligomers targetingvarious different regions of Enterococcus faecium 16S rRNA, designatedAVI-1-23-22,-32,-45,-33,-34, 44,-35 and -36 (SEQ ID NOS:92, 102, 115,103, 104, 114, 105, and 106), indicated in the figure as 22, 23, 45, 33,34, 44, 35 and 36, respectively, were added at 1 μM tovancomycin-resistant Enterococcus faecium (VRE) cultures, with theresults presented as percent viability;

FIG. 7 depicts the results of a study wherein PMO oligomers targetingvarious different regions of Enterococcus faecium 23S rRNA, designatedAVI-1-2346,-47, 48, 49 and -50 (SEQ ID NOS:116-120), indicated in thefigure as 46, 47, 48, 49 and 50, respectively, were added at 1 μM tovancomycin-resistant Enterococcus faecium cultures, with the resultspresented as percent viability;

FIG. 8 depicts the results of a study on the effect of 1 μM of PMOs ofvarious lengths targeted against the 16S rRNA of a vancomycin-resistantEnterococcus faecium bacterial strain on viability of the bacteria(percent viability, reported as percent of untreated control). The PMOsequences corresponding to the oligomer lengths are shown in Table 12,which illustrates antisense targeting of 16S rRNA in VRE, reported aspercent inhibition (100-percent of untreated control);

FIG. 9 depicts the results of a study on the effect of 1 μM PMO targetedagainst Enterococcus faecium 16S rRNA, designated VRE-2, AVI 1-23-22(SEQ ID NO:92), on bacterial colony formation in VRE, presented aspercent viability (percent of control) as determined on days 1 through6; and

FIGS. 10A-B depict the results of a study on the effect of 1 μM of a PMOtargeted against Enterococcus faecium 16S rRNA (SEQ ID NO:92), alone orin combination with (A) 3 μM vancomycin, or (B) 3 μM ampicillin, ongrowth of VRE, with the results reported as percent viability.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The terms below, as used herein, have the following meanings, unlessindicated otherwise:

As used herein, the term “16S ribosomal RNA”, also termed “16S rRNA”,refers to RNA which is part of the structure of a ribosome and isinvolved in the synthesis of proteins.

The term “polynucleotide” as used herein refers to a polymeric moleculehaving a backbone which supports bases capable of hydrogen bonding totypical polynucleotides, where the polymer backbone presents the basesin a manner to permit such hydrogen bonding in a sequence specificfashion between the polymeric molecule and a typical polynucleotide(e.g., single-stranded RNA, double-stranded RNA, single-stranded DNA ordouble-stranded DNA). “Polynucleotides” include polymers withnucleotides which are an N- or C-glycoside of a purine or pyrimidinebase, and polymers containing non-standard nucleotide backbones, forexample, backbones formed using phosphorodiamidate morpholino chemistry,polyamide linkages (e.g., peptide nucleic acids or PNAs) and othersynthetic sequence-specific nucleic acid molecules.

As used herein, the terms “antisense oligonucleotide” and “antisenseoligomer” are used interchangeably and refer to a sequence of nucleotidebases and a subunit-to-subunit backbone that allows the antisenseoligomer to hybridize to a target nucleic acid (e.g., RNA) sequence byWatson-Crick base pairing, to form a nucleic acid:oligomer heteroduplexwithin the target sequence. The oligomer may have exact sequencecomplementarity to the target sequence or near complementarity. In oneexemplary application, such an antisense oligomer may block or inhibitthe function of 16S or 23S rRNA containing a given target sequence, maybind to a double-stranded or single stranded portion of the 16S or 23SrRNA target sequence, may inhibit mRNA translation and/or proteinsynthesis, and may be said to be “directed to” a sequence with which itspecifically hybridizes.

As used herein, an oligonucleotide or antisense oligomer “specificallyhybridizes” to a target polynucleotide if the oligomer hybridizes to thetarget under physiological conditions, with a Tm substantially greaterthan 37° C., preferably at least 50° C., and typically 60° C.-80° C. orhigher. Such hybridization preferably corresponds to stringenthybridization conditions. At a given ionic strength and pH, the T_(m) isthe temperature at which 50% of a target sequence hybridizes to acomplementary polynucleotide.

Polynucleotides are described as “complementary” to one another whenhybridization occurs in an antiparallel configuration between twosingle-stranded polynucleotides. A double-stranded polynucleotide can be“complementary” to another polynucleotide, if hybridization can occurbetween one of the strands of the first polynucleotide and the second.Complementarity (the degree that one polynucleotide is complementarywith another) is quantifiable in terms of the proportion (i.e., thepercentage) of bases in opposing strands that are expected to formhydrogen bonds with each other, according to generally acceptedbase-pairing rules.

As used herein, the term “consensus sequence”, relative to 16S or 23SrRNA sequences, refers to a sequence which is common to or shared by aparticular group of organisms. The consensus sequence shows the nucleicacid most commonly found at each position within the polynucleotide. Forexample, a Gram-negative bacterial 16S or 23S rRNA consensus sequence iscommon to Gram-negative bacteria and generally not found in bacteriathat are not Gram-negative.

As used herein, the term “conserved”, relative to 16S or 23S rRNAsequences, also refers to a sequence which is common to or shared by aparticular group of organisms (e.g., bacteria).

A “subunit” of an oligonucleotide or oligonucleotide analog refers toone nucleotide (or nucleotide analog) unit of the oligomer. The term mayrefer to the nucleotide unit with or without the attached intersubunitlinkage, although, when referring to a “charged subunit”, the chargetypically resides within the intersubunit linkage (e.g. a phosphate orphosphorothioate linkage).

As used herein, a “morpholino oligomer” refers to a polymeric moleculehaving a backbone which supports bases capable of hydrogen bonding totypical polynucleotides, wherein the polymer lacks a pentose sugarbackbone moiety, and more specifically lacks a ribose backbone linked byphosphodiester bonds which is typical of nucleotides and nucleosides,but instead contains a ring nitrogen with coupling through the ringnitrogen. A typical “morpholino” oligonucleotide is composed ofmorpholino subunit structures of the form shown in FIGS. 1A-1D, where(i) the structures are linked together by phosphorous-containinglinkages, one to three atoms long, joining the morpholino nitrogen ofone subunit to the 5′ exocyclic carbon of an adjacent subunit, and (ii)P_(i) is a purine or pyrimidine base-pairing moiety effective to bind,by base-specific hydrogen bonding, to a base in a polynucleotide.

As used herein, the term “PMO” refers to a phosphorodiamidate morpholinooligomer, as further described below, wherein the oligomer is apolynucleotide of about 8-40 bases in length, preferably 12-25 bases inlength. This preferred aspect of the invention is illustrated in FIG.2B, where the two subunits are joined by a phosphorodiamidate linkage.

As used herein, a “nuclease-resistant” oligomeric molecule (oligomer) isone whose backbone is not susceptible to nuclease cleavage of aphosphodiester bond. Exemplary nuclease resistant antisense oligomersare oligonucleotide analogs such as phosphorothioate and phosphate-amineDNA (pnDNA), both of which have a charged backbone, and methylphosphonate and phosphoramidate- or phosphorodiamidate-linked morpholinooligonucleotides, which have uncharged backbones.

A “2′-O-allyl (or alkyl) modified oligonucleotide” is anoligoribonucleotide in which the 2′ hydroxyl is converted to an allyl oralkyl ether, respectively. The alkyl ether is typically a methyl ether.

“Alkyl” refers to a fully saturated acyclic monovalent radicalcontaining carbon and hydrogen, which may be branched or a straightchain. Examples of alkyl groups are methyl, ethyl, n-butyl, t-butyl,n-heptyl, and isopropyl. “Lower alkyl” refers to an alkyl radical of oneto six carbon atoms, and preferably one to four carbon atoms, asexemplified by methyl, ethyl, isopropyl, n-butyl, isobutyl, and t-butyl.

As used herein, a first sequence is an “antisense sequence” with respectto a second sequence if a polynucleotide with a first sequencespecifically binds to, or specifically hybridizes with, a polynucleotidewhich has a second sequence, under physiological conditions.

As used herein, a “base-specific intracellular binding event involving atarget RNA” refers to the specific binding of an oligomer to a targetRNA sequence inside a cell. The base specificity of such binding issequence specific. For example, a single-stranded polynucleotide canspecifically bind to a single-stranded polynucleotide that iscomplementary in sequence.

As used herein, “nuclease-resistant heteroduplex” refers to aheteroduplex formed by the binding of an antisense oligomer to itscomplementary target, such that the heteroduplex is resistant to in vivodegradation by ubiquitous intracellular and extracellular nucleases.

As used herein, the term “broad spectrum bacterial sequence”, withreference to bacterial 16S rRNA, refers to an oligonucleotide of theinvention which is antisense to some segment of most if not all of thebacterial 16S rRNA sequences described herein. A correspondingdefinition applies to bacterial 23S rRNA. Exemplary broad spectrumbacterial sequences described herein include the antisense oligomerspresented as SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, which areantisense to an Escherichia coli (E. coli), Salmonella thyphimurium (S.thyphi), Pseudomonas aeruginosa (P. aeruginosa), Vibrio cholera,Neisseria gonorrhoea (N. gonorrhoea), Staphylococcus aureus (Staph.aureus), Mycobacterium tuberculosis (Myco. tubercul.), Helicobacterpylori (H. pylori), Streptococcus pneumoniae (Strep. pneumoniae),Treponema palladium Treponema pallad.), Chlamydia trachomatis (Chlamydiatrach.), Bartonella henselae (Bartonella hens.), Hemophilis influenza(H. influenza) and Shigella dysenterae (Shigella dys.) 16S rRNA sequence(see Table 5A), and SEQ ID NOS:24-25, which are antisense to the 16srRNA of the majority of these organisms (see Table 5B).

As used herein, the term “narrow spectrum bacterial sequence”, withrespect to 16S bacterial rRNA, refers to an oligonucleotide of theinvention which is antisense to particular, but not most or all,bacterial 16S rRNA sequences described herein. Again, a correspondingdefinition applies to bacterial 23S rRNA. A narrow spectrum bacterialsequence may be specific to one or more different bacteria, e.g., anantisense oligomer which is antisense to E. coli, S. thyphi and Shigelladys. 16S rRNA, but not the other bacterial 16S rRNA sequences describedherein, as exemplified by SEQ ID NO:31; or an antisense oligomer whichis antisense to the E. coli 16S rRNA sequence, but not the otherbacterial 16S rRNA sequences described herein, as exemplified by SEQ IDNO:32.

As used herein, the term “modulating expression” relative tooligonucleotides refers to the ability of an antisense oligomer toeither enhance or reduce the expression of a given protein byinterfering with the expression or translation of RNA.

As used herein, “effective amount” relative to an antisense oligomerrefers to the amount of antisense oligomer administered to a mammaliansubject, either as a single dose or as part of a series of doses, thatis effective to inhibit a biological activity, e.g., expression of aselected target nucleic acid sequence.

As used herein, “treatment” of an individual or a cell is any type ofintervention provided as a means to alter the natural course of theindividual or cell. Treatment includes, but is not limited to,administration of a pharmaceutical composition, and may be performedeither prophylactically or subsequent to the initiation of a pathologicevent or contact with an etiologic agent.

As used herein, the term “improved therapeutic outcome”, relative to apatient diagnosed as infected with a particular bacteria, refers to aslowing or diminution in the growth of the bacteria and/or a decreasein, or elimination of, detectable symptoms typically associated withinfection by that particular bacteria.

II. Antisense Oligomers: Selection Criteria

Antisense compounds employed in the invention preferably meet severalcriteria of structure and properties, considered in the subsectionsbelow.

A. Base Sequence and Length

The antisense compound has a base sequence targeted against a selectedRNA target sequence. The region of complementarity with the target RNAsequence may be as short as 10-12 bases, but is preferably 13-20 bases,and more preferably 17-20 bases, in order to achieve the requisitebinding Tm, as discussed below.

In some cases, the target for modulation of the activity of 16S rRNAusing the antisense oligomers of the invention is a sequence in a doublestranded region of the 16s rRNA, such as the peptidyl transferasecenter, the alpha-sarcin loop or the mRNA binding region of the 16S rRNAsequence. In other cases, the target for modulation of gene expressionis a sequence in a single stranded region of bacterial 16S or 23S rRNA.The target may be a consensus sequence for bacterial 16S or 23S rRNAs ingeneral, a sequence common to the 16s or 23S rRNA of one or more typesof bacteria (e.g., Gram positive or Gram negative bacteria), or specificto a particular 16S or 23S rRNA sequence.

The oligomer may be 100% complementary to the bacterial RNA targetsequence, or it may include mismatches, e.g., to accommodate variants,as long as the heteroduplex formed between the oligomer and bacterialRNA target sequence is sufficiently stable to withstand the action ofcellular nucleases and other modes of degradation which may occur invivo. Mismatches, if present, are less destabilizing toward the endregions of the hybrid duplex than in the middle. The number ofmismatches allowed will depend on the length of the oligomer, thepercentage of G:C base pairs in the duplex and the position of themismatch(es) in the duplex, according to well understood principles ofduplex stability. Although such an antisense oligomer is not necessarily100% complementary to the bacterial RNA target sequence, it is effectiveto stably and specifically bind to the target sequence such that abiological activity of the nucleic acid target, e.g., expression ofbacterial protein(s) is modulated.

Oligomers as long as 40 bases may be suitable, where at least theminimum number of bases, e.g., 10-15 bases, are complementary to thetarget RNA sequence. In general, however, facilitated or active uptakein cells is optimized at oligomer lengths less than about 30, preferablyless than 25, and more preferably 20 or fewer bases. For PMO oligomers,described further below, an optimum balance of binding stability andintake generally occurs at lengths of 17-18 bases.

B. Duplex Stability (Tm)

The oligomer must form a stable hybrid duplex with the target sequence.Preferably, the oligomer is able to hybridize to the target RNA sequencewith a Tm substantially greater than the Tm of a duplex composed of acorresponding DNA and the same target RNA sequence. The antisenseoligomer will have a binding Tm, with respect to acomplementary-sequence RNA, of greater than body temperature andpreferably greater than 50° C. Tm's in the range 60-80° C. or greaterare preferred. The Tm of an antisense compound with respect tocomplementary-sequence RNA may be measured by conventional methods, suchas those described by Hames et al., Nucleic Acid Hybridization, IRLPress 1985, pp.107-108. According to well known principles, the Tm of anoligomer compound, with respect to a complementary-base RNA hybrid, canbe increased by increasing the length (in basepairs) of theheteroduplex. At the same time, for purposes of optimizing celltransport, it may be advantageous to limit the size of the oligomer. Forthis reason, compounds that show high Tm (50° C. or greater) at a lengthof 15-20 bases or less will be preferred over those requiring 20+ basesfor high Tm values.

Increasing the ratio of C:G paired bases in the duplex is also known togenerally increase in the Tm of an oligomer compound. Studies in supportof the invention suggest that maximizing the number of C bases in theantisense oligomer is particularly favorable.

C. Uptake by Cells

In order to achieve adequate intracellular levels, the antisenseoligomer must be actively taken up by cells, meaning that the compoundis taken up by facilitated or active transport, if administered in free(non-complexed) form, or is taken by an endocytotic mechanism ifadministered in complexed form.

When the antisense compound is administered in complexed form, thecomplexing agent typically is a polymer, e.g., a cationic lipid,polypeptide, or non-biological cationic polymer, having an oppositecharge to a net charge on the antisense compound. Methods of formingcomplexes, including bilayer complexes, between anionic oligonucleotidesand cationic lipid or other polymer components are well known. Forexample, the liposomal composition Lipofectin® (Felgner et al., 1987),containing the cationic lipid DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and theneutral phospholipid DOPE (dioleyl phosphatidyl ethanolamine), is widelyused. After administration, the complex is taken up by cells through anendocytotic mechanism, typically involving particle encapsulation inendosomal bodies. The ability of the antisense agent to resist cellularnucleases promotes survival and ultimate delivery of the agent to thecell cytoplasm.

In the case where the agent is administered in free form, the agentshould be substantially uncharged, meaning that a majority of itsintersubunit linkages are uncharged at physiological pH. Experimentscarried out in support of the invention indicate that a small number ofnet charges, e.g., 1-2 for a 15- to 20-mer oligomer, can enhance celluptake of certain oligomers with substantially uncharged backbones. Thecharges may be carried on the oligomer itself, e.g., in the backbonelinkages, or may be terminal charged-group appendages. Preferably, thenumber of charged linkages is no more than one charged linkage per fouruncharged linkages.

An oligomer may also contain both negatively and positively chargedbackbone linkages, as long as two opposite charges are substantiallyoffsetting, and preferably do not include runs of more than 3-5consecutive subunits of either charge. For example, the oligomer mayhave a given number of anionic linkages, e.g. phosphorothioate orN3′→P5′ phosphoramidate linkages, and a comparable number of cationiclinkages, such as N,N-diethylenediamine phosphoramidates (Dagle). Thenet charge is preferably neutral or at most 1-2 net charges peroligomer, as above.

In addition to being substantially or fully uncharged, the antisenseagent is preferably a substrate for a membrane transporter system (i.e.a membrane protein or proteins) capable of facilitating transport oractively transporting the oligomer across the cell membrane. Thisfeature may be determined by one of a number of tests, as follows, foroligomer interaction or cell uptake.

A first test assesses binding at cell surface receptors, by examiningthe ability of an oligomer compound to displace or be displaced by aselected charged oligomer, e.g., a phosphorothioate oligomer, on a cellsurface. The cells are incubated with a given quantity of test oligomer,which is typically fluorescently labeled, at a final oligomerconcentration of between about 10-300 nM. Shortly thereafter, e.g.,10-30 minutes (before significant internalization of the test oligomercan occur), the displacing compound is added, in incrementallyincreasing concentrations. If the test compound is able to bind to acell surface receptor, the displacing compound will be observed todisplace the test compound. If the displacing compound is shown toproduce 50% displacement at a concentration of 10× the test compoundconcentration or less, the test compound is considered to bind at thesame recognition site for the cell transport system as the displacingcompound.

A second test measures cell transport, by examining the ability of thetest compound to transport a labeled reporter, e.g., a fluorescencereporter, into cells. The cells are incubated in the presence of labeledtest compound, added at a final concentration between about 10-300 nM.After incubation for 30-120 minutes, the cells are examined, e.g., bymicroscopy, for intracellular label. The presence of significantintracellular label is evidence that the test compound is transported byfacilitated or active transport.

A third test relies on the ability of certain antisense compounds toeffectively inhibit bacterial growth when targeted against bacterial 16Sor 23S rRNA. Studies carried out in support of the present inventionshow that the inhibition requires active or facilitated transport acrossbacterial cell membranes. The test compound is prepared with a target16S sequence that has been demonstrated to be effective in inhibitingbacterial growth. For example, SEQ ID. NOS:1-3 herein are representativesequences against E. coli 16S rRNA. The compound is added to the growingbacterial culture at increasing concentrations, typically between 10 nMand 1 mM. The ability to inhibit bacterial growth is measured fromnumber of cell colonies cell counts at 24-72 hours after addition of thetest compound. Compounds which can produce a 50% inhibition at aconcentration of between about 100-500 nM or lower are considered to begood candidates for active transport.

As shown by the data in FIG. 4, 500 nM of PMO antisense oligomertargeted against VRE (vancomycin-resistant Enterococcus) 16s rRNA,having SEQ ID NO:92, inhibited growth in VRE by about 50%. It was alsoobserved that addition of a comparatively large concentration (50 μM) ofa nontarget sequence PMO (antisense to c-myc; SEQ ID NO:139) essentiallynullified this effect, suggesting that the transport mechanism has afinite capacity.

D. mRNA Resistance to RNaseH

Two general mechanisms have been proposed to account for inhibition ofexpression by antisense oligonucleotides. (See e.g., Agrawal et al.,1990; Bonham et al., 1995; and Boudvillain et al., 1997). In the first,a heteroduplex formed between the oligonucleotide and mRNA is asubstrate for RNaseH, leading to cleavage of the mRNA. Oligonucleotidesbelonging, or proposed to belong, to this class includephosphorothioates, phosphotriesters, and phosphodiesters (unmodified“natural” oligonucleotides). However, because such compounds wouldexpose mRNA in an oligomer:RNA duplex structure to proteolysis byRNaseH, and therefore loss of duplex, they are suboptimal for use in thepresent invention. A second class of oligonucleotide analogs, termed“steric blockers” or, alternatively, “RNaseH inactive” or “RNaseHresistant”, have not been observed to act as a substrate for RNaseH, andare believed to act by sterically blocking target RNA nucleocytoplasmictransport, splicing or translation. This class includesmethylphosphonates (Toulme et al., 1996), morpholino oligonucleotides,peptide nucleic acids (PNA's), 2′-O-allyl or 2′-O-alkyl modifiedoligonucleotides (Bonham, 1995), and N3′→P5′ phosphoramidates (Gee,1998; Ding).

A test oligomer can be assayed for its ability to protect mRNA againstRNaseH by forming an RNA:oligomer duplex with the test compound, thenincubating the duplex with RNaseH under a standard assay conditions, asdescribed in Stein et al. After exposure to RNaseH, the presence orabsence of intact duplex can be monitored by gel electrophoresis or massspectrometry.

In testing an oligomer for suitability in the present invention, each ofthe properties detailed above is preferably met. It is recognized thatthe “substantially uncharged” feature is inherently met where thelinkages are uncharged, and the target-sequence complementarity isachieved by base-sequence design. Thus, an oligomer is preferably testedas to its (i) Tm with respect to target RNA at a duplex lengthpreferably between 12-20 basepairs, (ii) ability to be transportedacross cell membranes by active or facilitated transport, and (iii)ability to prevent RNA proteolysis by RNaseH in duplex form.

The antibacterial effectiveness of a given antisense oligomer may befurther evaluated by screening methods known in the art. For example,the oligomer may be incubated with a bacterial culture in vitro and theeffect on the target 16S RNA evaluated by monitoring (1) heteroduplexformation with the target sequence and/or non-target sequences, usingprocedures known to those of skill in the art, e.g., an electrophoreticgel mobility assay; (2) the amount of 16S mRNA, as determined bystandard techniques such as RT-PCR or Northern blot; (3) the amount ofbacterial protein production, as determined by standard techniques suchas ELISA or Western blotting; or (4) the amount of bacterial growth invitro for both bacteria known to have the 16S rRNA sequence targeted bya particular antisense oligomer and bacteria not predicted to have thetarget 16S rRNA sequence.

Candidate antisense oligomers may also be evaluated, according to wellknown methods, for acute and chronic cellular toxicity, such as theeffect on protein and DNA synthesis as measured via incorporation of³H-leucine and ³H-thymidine, respectively. In addition, various controloligonucleotides, e.g., one or more control oligonucleotides such assense, nonsense or scrambled antisense sequences, or sequencescontaining mismatched bases, are generally included in the evaluationprocess, in order to confirm the specificity of binding of candidateantisense oligomers. The results of such tests allow discrimination ofspecific effects of antisense inhibition of gene expression fromindiscriminate suppression. (See, e.g. Bennett et al., 1995). Sequencesmay be modified as needed to limit non-specific binding of antisenseoligomers to non-target sequences, e.g., by changing the length or thedegree of complementarity to the target sequence.

III. Uncharged Oligonucleotide Analogs

Examples of uncharged linkages that may be used in oligonucleotideanalogs of the invention are shown in FIGS. 3A-3G. (As noted below, asmall number of charged linkages, e.g. charged phosphoramidate orphosphorothioate, may also be incorporated into the oligomers.) Theuncharged linkages include carbonate (3A, R═O) and carbamate (3A, R═NH₂)linkages, (Mertes; Gait); alkyl phosphonate and phosphotriester linkages(3B, R═alkyl or —O-alkyl) (Miller; Lesnikowski); amide linkages (3C);sulfones (3D, R₁, R₂═CH₂) (Roughten); sulfonamides (3D, R₁═NH, R₂═CH₂ orvice versa) (McElroy); sulfamates (3D, R₁, R₂═NH) (Huie); and athioformacetyl linkage (3E) (Matteucci; Cross). The latter is reportedto have enhanced duplex and triplex stability with respect tophosphorothioate antisense compounds (Cross). Also reported are the3′methylene-N-methylhydroxyamino compounds of structure 3F (Vasseur). InFIGS. 3A-3G, B represents a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, preferably selected from adenine, cytosine, guanine anduracil. The linkages join nucleotide subunits, each consisting of a 5-or 6-membered ring supporting a base-pairing moiety effective to bind byWatson-Crick base pairing to a respective nucleotide base in thebacterial nucleic acid sequence. These subunits may comprise, forexample, ribose rings, as in native nucleic acids, or morpholino rings,as described further below.

PNAs (peptide nucleic acids) are analogs of DNA in which the backbone isstructurally homomorphous with a deoxyribose backbone, consisting ofN-(2-aminoethyl) glycine units to which pyrimidine or purine bases areattached. PNAs containing natural pyrimidine and purine bases hybridizeto complementary oligonucleotides obeying Watson-Crick base-pairingrules, and mimic DNA in terms of base pair recognition (Egholm et al.,1993). However, PNA antisense agents have been observed to display slowmembrane penetration in cell cultures, possibly due to poor uptake(transport) into cells. (See, e.g., Ardhammar, M. et al., 1999).

Oligomeric ribonucleotides substituted at the 2′-oxygen show high RNAbinding affinities and, in comparison to unsubstituted ribonucleotides,reduced sensitivity to endogenous nucleases. Methyl-substitutedribonucleotides are reported to provide greater binding affinity andcellular uptake than those having larger 2′-oxygen substituents (e.g.ethyl, propyl, allyl, or pentyl).

One preferred oligomer structure employs morpholino-based subunitsbearing base-pairing moieties, joined by uncharged linkages as outlinedabove. Especially preferred is a substantially uncharged morpholinooligomer such as illustrated by the phosphorodiamidate-linked compoundshown in FIG. 3G. Morpholino oligonucleotides, including antisenseoligomers, are detailed, for example, in co-owned U.S. Pat. Nos.5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444,5,521,063, and 5,506,337, all of which are expressly incorporated byreference herein. Desirable chemical properties of the morpholino-basedsubunits are the ability to be linked in a oligomeric form by stable,uncharged backbone linkages, the ability of the polymer so formed tohybridize with a complementary-base target nucleic acid, includingtarget RNA, with high Tm, even with oligomers as short as 10-14 bases,the ability of the oligomer to be actively transported into mammaliancells, and the ability of the oligomer:RNA heteroduplex to resist RNAsedegradation.

Exemplary backbone structures for antisense oligonucleotides of theinvention include the morpholino subunit types shown in FIGS. 1A-D, eachlinked by an uncharged, phosphorous-containing subunit linkage. In thesefigures, the X moiety pendant from the phosphorous may be any of thefollowing: fluorine; an alkyl or substituted alkyl; an alkoxy orsubstituted alkoxy; a thioalkoxy or substituted thioalkoxy; or, anunsubstituted, monosubstituted, or disubstituted nitrogen, includingcyclic structures. Alkyl, alkoxy and thioalkoxy preferably include 1-6carbon atoms, and more preferably 1-4 carbon atoms. Monosubstituted ordisubstituted nitrogen preferably refers to lower alkyl substitution,and the cyclic structures are preferably 5- to 7-membered nitrogenheterocycles optionally containing 1-2 additional heteroatoms selectedfrom oxygen, nitrogen, and sulfur. Z is sulfur or oxygen, and ispreferably oxygen.

FIG. 1A shows a phosphorous-containing linkage which forms the five atomrepeating-unit backbone shown in FIG. 2A, where the morpholino rings arelinked by a 1-atom phosphoamide linkage.

Subunit B in FIG. 1B is designed for 6-atom repeating-unit backbones, asshown in FIG. 2B. In FIG. 1B, the atom Y linking the 5′ morpholinocarbon to the phosphorous group may be sulfur, nitrogen, carbon or,preferably, oxygen. The X and Z moieties are as defined above.Particularly preferred morpholino oligonucleotides include thosecomposed of morpholino subunit structures of the form shown in FIG. 2B,where X═NH₂ or N(CH₃)₂, Y═O, and Z═O.

Subunits C-D in FIGS. 1C-D are designed for 7-atom unit-length backbonesas shown for structures in FIGS. 2C and D. In Structure C, the X moietyis as in Structure B, and the moiety Y may be methylene, sulfur, orpreferably oxygen. In Structure D, the X and Y moieties are as inStructure B. In all subunits depicted in FIGS. 1 and 2, each Pi and Pjis a purine or pyrimidine base-pairing moiety effective to bind, bybase-specific hydrogen bonding, to a base in a polynucleotide, and ispreferably selected from adenine, cytosine, guanine and uracil.

As noted above, the substantially uncharged oligomer may advantageouslyinclude a limited number of charged linkages, e.g. up to about 1 perevery 5 uncharged linkages. In the case of the morpholino oligomers,such a charged linkage may be a linkage as represented by any of FIGS.2A-D, preferably FIG. 2B, where X is oxide (—O⁻) or sulfide (—S⁻).

The antisense compounds of the invention can be synthesized by stepwisesolid-phase synthesis, employing methods detailed in the referencescited above. The sequence of subunit additions will be determined by theselected base sequence (see Sections IID and IV below). In some cases,it may be desirable to add additional chemical moieties to the oligomercompounds, e.g. to enhance the pharmacokinetics of the compound or tofacilitate capture or detection of the compound. Such a moiety may becovalently attached, typically to the 5′- or 3′-end of the oligomer,according to standard synthesis methods. For example, addition of apolyethyleneglycol moiety or other hydrophilic polymer, e.g., one having10-100 polymer subunits, may be useful in enhancing solubility. One ormore charged groups, e.g., anionic charged groups such as an organicacid, may enhance cell uptake. A reporter moiety, such as fluorescein ora radiolabeled group, may be attached for purposes of detection.Alternatively, the reporter label attached to the oligomer may be aligand, such as an antigen or biotin, capable of binding a labeledantibody or streptavidin. In selecting a moiety for attachment ormodification of an oligomer antisense, it is generally of coursedesirable to select chemical compounds of groups that are biocompatibleand likely to be tolerated by a subject without undesirable sideeffects.

IV. Exemplary Bacterial Targets

Escherichia coli (E. coli) is a Gram negative bacteria that is part ofthe normal flora of the gastrointestinal tract. There are hundreds ofstrains of E. coli, most of which are harmless and live in thegastrointestinal tract of healthy humans and animals. Currently, thereare four recognized classes of enterovirulent E. coli (the “EEC group”)that cause gastroenteritis in humans. Among these are theenteropathogenic (EPEC) strains and those whose virulence mechanism isrelated to the excretion of typical E. coli enterotoxins. Such strainsof E. coli can cause various diseases including those associated withinfection of the gastrointestinal tract and urinary tract, septicemia,pneumonia, and meningitis. Antibiotics are not effective against somestrains and do not necessarily prevent recurrence of infection.

For example, E. coli strain 0157:H7 is estimated to cause 10,000 to20,000 cases of infection in the United States annually (Federal Centersfor Disease Control and Prevention). Hemorrhagic colitis is the name ofthe acute disease caused by E. coli 0157:H7. Preschool children and theelderly are at the greatest risk of serious complications. E. colistrain 0157:H7 was recently reported as the cause of death of fourchildren who ate under cooked hamburgers from a fast-food restaurant inthe Pacific Northwest.

Salmonella thyphimurium are Gram negative bacteria which cause variousconditions that range clinically from localized gastrointestinalinfections and gastroenterits (diarrhea, abdominal cramps, and fever) toenteric fevers (including typhoid fever) which are serious systemicillnesses. Salmonella infection also causes substantial losses oflivestock.

Typical of Gram-negative bacilli, the cell wall of Salmonella spp.contains a complex lipopolysaccharide (LPS) structure that is liberatedupon lysis of the cell and may function as an endotoxin, whichcontributes to the virulence of the organism.

Contaminated food is the major mode of transmission for non-typhoidalsalmonella infection, due to the fact that Salmonella survive in meatsand animal products that are not thoroughly cooked. The most commonanimal sources are chickens, turkeys, pigs, and cows, in addition tonumerous other domestic and wild animals. The epidemiology of typhoidfever and other enteric fevers caused by Salmonella spp. is associatedwith water contaminated with human feces.

Vaccines are available for typhoid fever and are partially effective;however, no vaccines are available for non-typhoidal Salmonellainfection. Non-typhoidal salmonellosis is controlled by hygienicslaughtering practices and thorough cooking and refrigeration of food.Antibiotics are indicated for systemic disease, and Ampicillin has beenused with some success. However, in patients under treatment withexcessive amounts of antibiotics, patients under treatment withimmunosuppressive drugs, following gastric surgery, and in patients withhemolytic anemia, leukemia, lymphoma, or AIDS, Salmonella infectionremains a medical problem.

Pseudomonas spp. are motile, Gram-negative rods which are clinicallyimportant because they are resistant to most antibiotics, and are amajor cause of hospital acquired (nosocomial) infections. Infection ismost common in: immunocompromised individuals, burn victims, individualson respirators, individuals with indwelling catheters, IV narcotic usersand individuals with chronic pulmonary disease (e.g., cystic fibrosis).Although infection is rare in healthy individuals, it can occur at manysites and lead to urinary tract infections, sepsis, pneumonia,pharyngitis, and numerous other problems, and treatment often fails withgreater significant mortality.

Vibrio cholerae is a Gram negative rod which infects humans and causescholera, a disease spread by poor sanitation, resulting in contaminatedwater supplies. Vibrio cholerae can colonize the human small intestine,where it produces a toxin that disrupts ion transport across the mucosa,causing diarrhea and water loss. Individuals infected with Vibriocholerae require rehydration either intravenously or orally with asolution containing electrolytes. The illness is generallyself-limiting; however, death can occur from dehydration and loss ofessential electrolytes. Antibiotics such as tetracycline have beendemonstrated to shorten the course of the illness, and oral vaccines arecurrently under development.

Neisseria gonorrhoeae is a Gram negative coccus, which is the causativeagent of the common sexually transmitted disease, gonorrhea. Neisseriagonorrhoeae can vary its surface antigens, preventing development ofimmunity to reinfection. Nearly 750,000 cases of gonorrhea are reportedannually in the United States, with an estimated 750,000 additionalunreported cases annually, mostly among teenagers and young adults.Ampicillin, amoxicillin, or some type of penicillin used to berecommended for the treatment of gonorrhea. However, the incidence ofpenicillin-resistant gonorrhea is increasing, and new antibiotics givenby injection, e.g., ceftriaxone or spectinomycin, are now used to treatmost gonococcal infections.

Staphylococcus aureus is a Gram positive coccus which normally colonizesthe human nose and is sometimes found on the skin. Staphylococcus cancause bloodstream infections, pneumonia, and surgical-site infections inthe hospital setting (i.e., nosocomial infections). Staph. aureus cancause severe food poisoning, and many strains grow in food and produceexotoxins. Staphylococcus resistance to common antibiotics, e.g.,vancomycin, has emerged in the United States and abroad as a majorpublic health challenge both in community and hospital settings.Recently a vancomycin-resistant Staph. aureus isolate has also beenidentified in Japan.

Mycobacterium tuberculosis is a Gram positive bacterium which is thecausative agent of tuberculosis, a sometimes crippling and deadlydisease. Tuberculosis is on the rise globally and is the leading causeof death from a single infectious disease (with a current death rate ofthree million people per year). It can affect several organs of thehuman body, including the brain, the kidneys and the bones; however,tuberculosis most commonly affects the lungs.

In the United States, approximately ten million individuals are infectedwith Mycobacterium tuberculosis, as indicated by positive skin tests,with approximately 26,000 new cases of active disease each year. Theincrease in tuberculosis (TB) cases has been associated with HIV/AIDS,homelessness, drug abuse and immigration of persons with activeinfections. Current treatment programs for drug-susceptible TB involvetaking two or four drugs (e.g., isoniazid, rifampin, pyrazinamide,ethambutol or streptomycin) for a period of from six to nine months,because all of the TB germs cannot be destroyed by a single drug. Inaddition, the observation of drug-resistant and multiple drug resistantstrains of Mycobacterium tuberculosis is on the rise.

Helicobacter pylori (H. pylori) is a micro-aerophilic, Gram negative,slow-growing, flagellated organism with a spiral or S-shaped morphologywhich infects the lining of the stomach. H. pylori is a human gastricpathogen associated with chronic superficial gastritis, peptic ulcerdisease, and chronic atrophic gastritis leading to gastricadenocarcinoma. H. pylori is one of the most common chronic bacterialinfections in humans and is found in over 90% of patients with activegastritis. Current treatment includes triple drug therapy with bismuth,metronidazole, and either tetracycline or amoxicillin, which eradicatesH. pylori in most cases. Problems with triple therapy include patientcompliance, side effects, and metronidazole resistance. Alternateregimens of dual therapy which show promise are amoxicillin plusmetronidazole or omeprazole plus amoxicillin.

Streptococcus pneumoniae is a Gram positive coccus and one of the mostcommon causes of bacterial pneumonia as well as middle ear infections(otitis media) and meningitis. Each year in the United States,pneumococcal diseases account for approximately 50,000 cases ofbacteremia; 3,000 cases of meningitis; 100,000-135,000 hospitalizations;and 7 million cases of otitis media. Pneumococcal infection causes anestimated 40,000 deaths annually in the United States. Children lessthan 2 years of age, adults over 65 years of age, persons of any agewith underlying medical conditions, including, e.g., congestive heartdisease, diabetes, emphysema, liver disease, sickle cell, HIV, and thoseliving in special environments, e.g., nursing homes and long-term carefacilities, are at highest risk for infection.

Drug-resistant S. pneumoniae strains have become common in the UnitedStates, with many penicillin-resistant pneumococci also resistant toother antimicrobial drugs, such as erythromycin ortrimethoprim-sulfamethoxazole.

Treponema palladium is a spirochete which causes syphilis. T. pallidumis exclusively a pathogen which causes syphilis, yaws and non-venerealendemic syphilis or pinta. Treponema pallidum cannot be grown in vitroand does replicate in the absence of mammalian cells. The initialinfection causes an ulcer at the site of infection; however, thebacteria move throughout the body, damaging many organs over time. Inits late stages, untreated syphilis, although not contagious, can causeserious heart abnormalities, mental disorders, blindness, otherneurologic problems, and death.

Syphilis is usually treated with penicillin, administered by injection.Other antibiotics are available for patients allergic to penicillin, orwho do not respond to the usual doses of penicillin. In all stages ofsyphilis, proper treatment will cure the disease, but in late syphilis,damage already done to body organs cannot be reversed.

Chlamydia trachomatis is the most common bacterial sexually transmitteddisease in the United States, and it is estimated that 4 million newcases occur each year. The highest rates of infection are in 15 to 19year olds. Chlamydia is a major cause of non-gonococcal urethritis(NGU), cervicitis, bacterial vaginitis, and pelvic inflammatory disease(PID). Chlamydia infections may have very mild symptoms or no symptomsat all; however, if left untreated, Chlamydia infections can lead toserious damage to the reproductive organs, particularly in women.Antibiotics such as azithromycin, erythromycin, oflloxacin, amoxicillinor doxycycline are typically prescribed to treat Chlamydia infection.

Bartonella henselae. Cat Scratch Fever (CSF) or cat scratch disease(CSD) is a disease of humans acquired through exposure to cats, causedby a Gram negative rod originally named Rochalimaea henselae, andcurrently known as Bartonella henselae. Symptoms include fever andswollen lymph nodes. CSF is generally a relatively benign, self-limitingdisease in people; however, infection with Bartonella henselae canproduce distinct clinical symptoms in immunocompromised people,including acute febrile illness with bacteremia, bacillary angiomatosis,peliosis hepatis, bacillary splenitis, and other chronic diseasemanifestations such as AIDS encephalopathy.

The disease is treated with antibiotics, such as doxycycline,erythromycin, rifampin, penicillin, gentamycin, ceftriaxone,ciprofloxacin, and azithromycin.

Haemophilus influenzae (H. influenza) is a family of Gram negativebacteria; six types of which are known, with most H. influenza-relateddisease caused by type B, or “HIB”. Until a vaccine for HIB wasdeveloped, HIB was a common causes of otitis media, sinus infections,bronchitis, the most common cause of meningitis, and a frequent culpritin cases of pneumonia, septic arthritis (joint infections), cellulitis(infections of soft tissues), and pericarditis (infections of themembrane surrounding the heart). The H. influenza type B bacterium iswidespread in humans and usually lives in the throat and nose withoutcausing illness. Unvaccinated children under age 5 are at risk for HIBdisease. Meningitis and other serious infections caused by H. influenzainfection can lead to brain damage or death.

Shigella dysenteriae (Shigella dys.) is a Gram negative rod which causesdysentary. In the colon, the bacteria enter mucosal cells and dividewithin mucosal cells, resulting in an extensive inflammatory response.Shigella infection can cause severe diarrhea which may lead todehydration and can be dangerous for the very young, very old orchronically ill. Shigella dys. forms a potent toxin (shiga toxin), whichis cytotoxic, enterotoxic, and neurotoxic and acts as a inhibitor ofprotein synthesis. Resistance to antibiotics such as ampicillin andTMP-SMX has developed; however, treatment with newer, more expensiveantibiotics such as ciprofloxacin, norfloxacin and enoxacin, remainseffective.

Enterococcus faecium. Enterococci are a component of the normal flora ofthe gastrointestinal and female urogenital tracts; however, recentstudies indicate that pathogenic Enterococci can be transmitted directlyin the hospital setting. (See, e.g., Boyce et al., J Clin Microbiol 32,1148-53, 1994.) Enterococci have been recognized as a cause ofnosocomial infection and some strains are resistant to multipleantimicrobial drugs. The most common Enterococci-associated nosocomialinfections are urinary tract infections, post-surgical infections andbacteremia (Murray B E, Clin Microbiol 3, 46-65, Rev. 1990; Moellering RC Jr., Clin Infect Dis 14, 1173-8, 1992; Schaberg DR et al., Am J Med91(Suppl 3B), 72S-75S, 1991).

Vancomycin has been used extensively to treat Enterococcus infectionsince the late 1970s. Recently, a rapid increase in the incidence ofinfection and colonization with vancomycin-resistant enterococci (VRE)has been reported. The observed resistance is of concern due to (1) thelack of effective antimicrobial therapy for VRE infections because mostVRE are also resistant to drugs previously used to treat suchinfections, i. e., penicillin and aminoglycosides (CDC. MMWR 42:597-9,1993; Handwerger, et al., Clin Infect Dis 16, 750-5, 1993); and (2) thepossibility that the vancomycin-resistant genes present in VRE can betransferred to other gram-positive microorganisms.

Resistance to vancomycin and other glycopeptide antibiotics has beenassociated with the synthesis of a modified cell-wall precursor,terminating in D-lactate which has a lower affinity for antibiotics suchas vancomycin.

Listeria is a genus of Gram-positive, motile bacteria found in human andanimal feces. Listeria monocytogenes causes such diseases asmeningoencephalitis and meningitis. In cattle and sheep, listeriainfection causes encephalitis and spontaneous abortion.

Veterinary applications. A healthy microflora in the gastro-intestinaltract of livestock is of vital importance for health and correspondingproduction of associated food products. As with humans, thegastrointestinal tract of a healthy animal contains numerous types ofbacteria (i.e., E. coli, Pseudomonas aeruginosa and Salmonella spp.),which live in ecological balance with one another. This balance may bedisturbed by a change in diet, stress, or in response to antibiotic orother therapeutic treatment, resulting in bacterial diseases in theanimals generally caused by bacteria such as Salmonella, Campylobacter,Enterococci, Tularemia and E. coli. Bacterial infection in these animalsoften necessitates therapeutic intervention, which has treatment costsas well being frequently associated with a decrease in productivity.

As a result, livestock are routinely treated with antibiotics tomaintain the balance of flora in the gastrointestinal tract. Thedisadvantages of this approach are the development of antibioticresistant bacteria and the carry over of such antibiotics into resultingfood products.

V. Exemplary 16S rRNA Antisense Oligomers

In one embodiment, the antisense oligomers of the invention are designedto hybridize to a region of a bacterial 16S rRNA nucleic acid sequenceunder physiological conditions, with a T_(m) substantially greater than37° C., e.g., at least 50° C. and preferably 60° C. -80° C. The oligomeris designed to have high binding affinity to the nucleic acid and may be100% complementary to the 16S rRNA nucleic acid target sequence, or itmay include mismatches, as further described above.

In various aspects, the invention provides an antisense oligomer havinga nucleic acid sequence effective to stably and specifically bind to atarget sequence selected from the group consisting of 16S rRNA sequenceswhich have one or more of the following characteristics: (1) a sequencefound in a double stranded region of a 16s rRNA, e.g., the peptidyltransferase center, the alpha-sarcin loop and the mRNA binding region ofthe 16S rRNA sequence; (2) a sequence found in a single stranded regionof a bacterial 16s rRNA; (3) a sequence specific to a particular strainof a given species of bacteria, i.e., a strain of E. coli associatedwith food poisoning; (4) a sequence specific to a particular species ofbacteria; (5) a sequence common to two or more species of bacteria; (6)a sequence common to two related genera of bacteria (i. e., bacterialgenera of similar phylogenetic origin); (7) a sequence generallyconserved among Gram-negative bacterial 16S rRNA sequences; (6) asequence generally conserved among Gram-positive bacterial 16S rRNAsequences; or (7) a consensus sequence for bacterial 16S rRNA sequencesin general.

Exemplary bacteria and associated GenBank Accession Nos. for 16S rRNAsequences are provided in Table 1, below.

TABLE 1 GenBank Reference Organism for 16S rRNA SEQ ID NO: Escherichiacoli X80725 1 Salmonella thyphimurium U88545 2 Pseudomonas aeruginosaAF170358 3 Vibrio cholera AF118021 4 Neisseria gonorrhoea X07714 5Staphylococcus aureus Y15856 6 Mycobacterium tuberculosis X52917 7Helicobacter pylori M88157 8 Streptococcus pneumoniae AF003930 9Treponema palladium AJ010951 10 Chlamydia trachomatis D85722 11Bartonella henselae X89208 12 Hemophilis influenza M35019 13 Shigelladysenterae X96966 14

It will be understood that one of skill in the art may readily determineappropriate targets for antisense oligomers, and design and synthesizeantisense oligomers using techniques known in the art. Targets can beidentified by obtaining the sequence of a target 16S or 23S nucleic acidof interest (e.g. from GenBank) and aligning it with other 16S or 23Snucleic acid sequences using, for example, the MacVector 6.0 program, aClustalW algorithm, the BLOSUM 30 matrix, and default parameters, whichinclude an open gap penalty of 10 and an extended gap penalty of 5.0 fornucleic acid alignments. An alignment may also be carried out using theLasergene99 MegAlign Multiple Alignment program with a ClustalWalgorithm run under default parameters.

For example, given the 16s rRNA sequences provided in Table 1 and other16s rRNA sequences available in GenBank, one of skill in the art canreadily align the 16s rRNA sequences of interest and determine whichsequences are conserved among one or more different bacteria, and thosewhich are specific to one or more particular bacteria. A similaralignment can be performed on 23S rRNA sequences.

As an illustration, the 16S rRNA sequences from the organisms shown inTable 1 were aligned using the Lasergene 99 MegAlign Multiple Alignmentprogram, with a ClustalW algorithm and default parameters. Tables 2-5show exemplary oligomers antisense to 16S rRNA of these bacterialspecies, including sequences targeting individual bacteria, multiplebacteria, and broad spectrum sequences. These oligomers were derivedfrom the sequences in Table 1 and from the alignment performed asdescribed above. As the Tables show, a number of sequences wereconserved among different organisms.

Exemplary oligomers antisense to E. coli 16S rRNA (SEQ ID NO:32 and SEQID NO:35) were designed based on the sequence found at GenBank AccessionNo. X80725. Further exemplary oligomers antisense to E. coli 16S rRNAand one or more other bacterial 16S rRNA sequences are provided in Table2A.

Exemplary oligomers antisense to Salmonella thyphimurium 16S rRNA (SEQID NO:18 and SEQ ID NO:36) were designed based on the sequence found atGenBank Accession No. U88545.

Further exemplary oligomers antisense to S. thyphi. 16S rRNA and one ormore other bacterial 16S rRNA sequences are provided in Table 2A.

Exemplary oligomers antisense to Pseudomonas aeruginosa 16S rRNA (SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43) were designed basedon the sequence found at GenBank Accession No. AF170358.

Exemplary oligomers antisense to Vibrio cholera 16S rRNA (SEQ ID NO:45,SEQ ID NO:46 and SEQ ID NO:47) were designed based on the sequence foundat GenBank Accession No. AF118021. A further exemplary oligomer,antisense to Vibrio cholera 16S rRNA and other bacterial 16S rRNAsequences (SEQ ID NO:44), is provided in Table 2A.

TABLE 2A BACTERIAL 16s rRNA SEQUENCES AND ANTISENSE OLIGOMERS GenBankOrganism Reference Native sequence Antisense oligomer E. coli X80725 ntGAGTAAAGTTAAT GCAAAGGTATTAA (NS-1) 446-466 Shigella dys. X96966 ntACCTTTGC CTTTACT 436-456 (SEQ ID NO: 17) E. coli X80725 nt TCATAAAGTGCGTGGACTACGACGCA (BS-1) 1270- 1290 S. thyphi U88545 nt CGTAGTCC CTTTATGAG1282- 1302 Shigella dys. X96966 nt (SEQ ID NO: 15) 1263- 1283 E. coliX80725 nt AGTTTGATCATGG AATCTGAGCCATG 1-21 S. thyphi U88545 nt CTCAGATTATCAAACT 10-30 H. influenza M35019 (SEQ ID NO: 31) nt 10-30 E. coliX80725 nt ACGTCGCAAGCAC CCCTCTTTGTGCT 173-193 AAAGAGGG TGCGACGT (SEQ IDNO: 32) E. coli X80725 nt TTGAGTCTCGTAG ACCCCCCTCTACG 643-663 S. thyphiU88545 nt AGGGGGGT AGACTCAA 652-672 Shigella dys. X96966 nt (SEQ ID NO:33) 653-673 E. coli X80725 nt GGTTGTGCCCTTG CCACGCCTCAAGG 823-843 S.thyphi U88545 nt AGGCGTGG GCACAACC 832-852 Shigella dys. X96966 nt (SEQID NO: 34) 813-833 E. coli X80725 nt CGGAAGTTTTCAG TCTCATCTCTGAA991-1011 AGATGAGA AACTTCCG (SEQ ID NO: 35) S. thyphi U88545 ntGTTGTGGTTAATA GCTGCGGTTATTA (NS-2) 455-475 ACCGCAGC ACCACAAC (SEQ ID NO:18) S. thyphi. U88545 nt CCTCGCGAGAGCA GGTCCGCTTGCTC (BS-2) 1261- 1281E. coli X80725 nt AGCGGACC TCGCGAGG 1252- 1272 Shigella dys. X96966 nt(SEQ ID NO: 16) 1242- 1262 S. thyphi. U88545 nt AAATTGAAGAGTTCATGATCAAACTC 1-21 TGATCATG TTCAATTT (SEQ ID NO: 36) S. thyphi. U88545nt ACGTCGCAAGACC CCCTCTTTGGTCT 181-201 Shigella dys. X96966 nt AAAGAGGGTGCGACGT 162-182 (SEQ ID NO: 37) S. thyphi. U88545 nt TGAGTCTCGTAGATACCCCCCTCTAC 652-672 E. coli X80725 nt GGGGGGTA GAGACTCA 643-663Shigella dys. X96966 nt (SEQ ID NO: 38) 633-653 S. thyphi. U88545 ntGTTGTGCCCTTGA GCCACGCCTCAAG 832-852 E. coli X80725 nt GGCGTGGC GGCACAAC823-843 Shigella dys. X96966 nt (SEQ ID NO: 39) 813-833 P. AF170358ATGAAGAGGGCTT CAGAGAGCAAGC aeruginosa nt 1-21 GCTCTCTG CCTCTTCAT (SEQ IDNO: 40) P. AF170358 CGTCCTACGGGAG CCTGCTTTCTCCC aeruginosa nt 107-AAAGCAGG GTAGGACG 127 (SEQ ID NO: 41) P. AF170358 AGAGTATGGCAGACACCACCCTCTGC aeruginosa nt 578- GGGTGGTG CATACTCT 598 (SEQ ID NO: 42)P. AF170358 TTGGGATCCTTGA CTAAGATCTCAAG aeruginosa nt 758- GATCTTAGGATCCCAA 778 (SEQ ID NO: 43) Vibrio AF118021 ATTGAACGCTGGC GGCCTGCCGCCAGcholera nt 1-21 E. coli X80725 nt GGCAGGCC CGTTCAAT 19-39 (SEQ ID NO:44) H. influenza M35019 nt 26-46 S. thyphi. U88545 nt 18-48 ShigellaX96966 nt dys. 9-29 Vibrio AF118021 ATGTTTACGGACC CCCTCTTTGGTCC cholerant 157- AAAGAGGG GTAAACAT 177 (SEQ ID NO: 45) Vibrio AF118021GCTAGAGTCTTGT CCCCCTCTACAAG cholera nt 625- AGAGGGGG ACTCTAGC 645 (SEQID NO: 46) Vibrio AF118021 GAGGTTGTGACCT ACGACTYTAGGTC cholera nt 805-ARAGTCGT ACAACCTC 825 (SEQ ID NO: 47) 1: Approximate nucleotidelocations

Exemplary oligomers antisense to Neisseria gonorrhoea 16S rRNA (SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51) were designed basedon the sequence found at GenBank Accession No. X07714. These are shownin Table 2B, below.

Exemplary oligomers antisense to Staphylococcus aureus 16S rRNA (SEQ IDNO:53, SEQ ID NO:54 and SEQ ID NO:55) were designed based on thesequence found at GenBank Accession No. Y15856. A further exemplaryoligomer, antisense to a Staph. aureus 16S rRNA and a Bartonellahenselae 16S rRNA sequence (SEQ ID NO:52), is provided in Table 2B,below.

Exemplary oligomers antisense to Mycobacterium tuberculosis 16S rRNA(SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59) weredesigned based on the sequence found at GenBank Accession No. X52917.

Exemplary oligomers antisense to Helicobacter pylori 16S rRNA (SEQ IDNO:60, SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63) were designed basedon the sequence found at GenBank Accession No. M88157.

Exemplary oligomers antisense to Streptococcus pneumoniae 16S rRNA (SEQID NO:64, SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:67) were designedbased on the sequence found at GenBank Accession No. AF003930.

Exemplary oligomers antisense to Treponema palladium 16S rRNA (SEQ IDNO:69, SEQ ID NO:70 and SEQ ID NO:71) were designed based on thesequence found at GenBank Accession No. AJ010951. A further exemplaryoligomer, antisense to Treponema palladium 16S rRNA and other 16S rRNAsequences (SEQ ID NO:68), is provided in Table 2B, below.

TABLE 2B BACTERIAL 16s rRNA SEQUENCES AND ANTISENSE OLIGOMERS GenBankOrganism Reference Native sequence Antisense oligomer N. gonorrhoeaX07714 TGAACATAAGAGT AGGATCAAAC- nt 1-21 TTGATCCT TCTTATGTTCA (SEQ IDNO: 48) N. gonorrhoea X07714 CGTCTTGAGAGGG CCTGCTTTCC- nt 183- AAAGCAGGCTCTCAAGACG 203 (SEQ ID NO: 49) N. gonorrhoea X07714 CGAGTGTGTCAGACACCTCCCTC- nt 654- GGGAGGTG TGACACACTCG 674 (SEQ ID NO: 50) N.gonorrhoea X07714 TTGGGCAACTTGA CCAAGCAATC- nt 834- TTGCTTGG AAGTTGCCCAA854 (SEQ ID NO: 51) Staph. aureus Y15856 CTGGCTCAGGATG CCAGCGTTCA- nt1-21 AACGCTGG TCCTGAGCCAG (SEQ ID NO: 52) Bartonella hens X89208 nt 3-23Staph. aureus Y15856 ATATTTTGAACCG GAACCATGCG- nt 163- CATGGTTCGTTCAAAATAT 183 (SEQ ID NO: 53) Staph. aureus Y15856 CTTGAGTGCAGAACTTTCCTCTT- nt 640- GAGGAAAG CTGCACTCAAG 660 (SEQ ID NO: 54) Staph.aureus Y15857 ATGTGCACAG- nt 447- TTACTTACAC 466 avi ref no. 23 Staph.aureus Y15857 CTGAGAACAA- nt 1272- CTTTATGGGA 1291 avi ref no. 24 Staph.aureus Y15856 GTGTTAGGGGGTT GGGGCGGAAA- nt 819- TCCGCCCC CCCCCTAACAC 839(SEQ ID NO: 55) Myco. tubercul. X52917 GGCGGCGTGCTTA GCATGTGTTA- nt 1-21ACACATGC AGCACGCCGCC (SEQ ID NO: 56) Myco. tubercul. X52917GGACCACGGGATG AAGACATGCA- nt 138- CATGTCTT TCCCGTGGTCC 158 (SEQ ID NO:57) Myco. tubercul. X52917 AGAGTACTGCAGG CAGTCTCCCC- nt 604- GGAGACTGTGCAGTACTCT 624 (SEQ ID NO: 58) Myco. tubercul. X52917 TGGGTTTCCTTCCTGATCCCAAGG- nt 784- TGGGATC AAGGAAACCCA 804 (SEQ ID NO: 59) H. pyloriM88157 TTTATGGAGAGTT CAGGATCAAA- nt 1-21 TGATCCTG CTCTCCATAAA (SEQ IDNO: 60) H. pylori M88157 ACTCCTACGGGGG AAATCTTTCC- nt 181- AAAGATTTCCCGTAGGAGT 201 (SEQ ID NO: 61) H. pylori M88157 AGAGTGTGGGAGACACCTACCTC- nt 613- GGTAGGTG TCCCACACTCT 633 (SEQ ID NO: 62) H. pyloriM88157 TTGGAGGGCTTAG TGGAGAGACT- nt 794- TCTCTCCA AAGCCCTCCAA 814 (SEQID NO: 63) Strep. AF003930 ATTTGATCCTGGC CGTCCTGAGC- pneumoniae nt 1-21TCAGGACG CAGGATCAAAT (SEQ ID NO: 64) Strep. AF003930 AGAGTGGATGTTGATGTCATGCA- pneumoniae 169- CATGACAT ACATCCACTCT 189 (SEQ ID NO: 65)Strep. AF003930 TTGAGTGCAAGAG ACTCTCCCCT- pneumoniae 646- GGGAGAGTCTTGCACTCAA 666 (SEQ ID NO: 66) Strep. AF003930 GTTAGACCCTTTCAAACCCCGGA- pneumoniae 826- CGGGGTTT AAGGGTCTAAC 846 (SEQ ID NO: 67)Treponema AJ010951 AGAGTTTGATCAT TCTGAGCCAT- pallad. nt 1-21 GGCTCAGAGATCAAACTCT (SEQ ID NO: 68) S. thyphi. U88545 nt 8-28 H. influenzaM35019 nt 8-28 Treponema AJ010951 ACTCAGTGCTTCA ACCCCTTATG- pallad. nt173- TAAGGGGT AAGCACTGAGT 193 (SEQ ID NO: 69) Treponema AJ010951TTGAATTACGGAA AGTTTCCCTT- pallad. nt 651- GGGAAACT CCGTAATTCAA 671 (SEQID NO: 70) Treponema AJ010951 GTTGGGGCAAGAG CACTGAAGCT- pallad. nt 831-CTTCAGTG CTTGCCCCAAC 851 (SEQ ID NO: 71) 2 Approximate nucleotidelocations

Exemplary oligomers antisense to Chlamydia trachomatis 16S rRNA (SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75) were designed basedon the sequence found at GenBank Accession No. D85722. These are shownin Table 2C, below.

Exemplary oligomers antisense to Bartonella henselae 16S rRNA (SEQ IDNO:76, SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79) were designed basedon the sequence found at GenBank Accession No. X89208.

Exemplary oligomers antisense to Hemophilis influenza 16S rRNA (SEQ IDNO:81, SEQ ID NO:82 and SEQ ID NO:83) were designed based on thesequence found at GenBank Accession No. M35019. A further exemplaryoligomer, antisense to a H. influenza 16S rRNA sequence and a Salmonellathyphimurium 16S rRNA sequence (SEQ ID NO:80), is provided in Table 2C,below.

An exemplary oligomer antisense to Shigella dysenterae 16S rRNA (SEQ IDNO:88) was designed based on the sequence found at GenBank Accession No.X96966. Further exemplary antisense oligomers antisense to Shigella dys16S rRNA and one or more other bacterial 16S rRNA sequences are providedin Table 2C.

TABLE 2C BACTERIAL 16s rRNA SEQUENCES AND ANTISENSE OLIGOMERS GenBankOrganism Reference Native sequence Antisense oligomer Chlamydia D85722CTGAGAATTTGA GAACCAAGAT- trach. nt 1-21 TCTTGGTTC CAAATTCTCAG (SEQ IDNO: 72) Chlamydia D85722 ATATTTGGGCATC GTTACTCGGA- trach. nt 176-CGAGTAAC TGCCCAAATAT 196 (SEQ ID NO: 73) Chlamydia D85722 AGAGGGTAGATGCCTTTTCTCC- trach. nt 658- GAGAAAAGG ATCTACCCTCT 678 (SEQ ID NO: 74)Chlamydia D85722 TGGATGGTCTCA GGATGGGGTTG- trach. nt 838- ACCCCATCCAGACCATCCA 858 (SEQ ID NO: 75) Bartonella X89208 TCCTGGCTCAGGAGCGTTCATC- hens. nt 1-21 ATGAACGCT CTGAGCCAGGA (SEQ ID NO: 76)Bartonella X89208 CGTCCTACTGGA AAATCTTTCT- hens. nt 149- GAAAGATTTCCAGTAGGACG 169 (SEQ ID NO: 77) Bartonella X89208 TGAGTATGGAAGCACTCACCTC- hens. nt 581- AGGTGAGTG TTCCATACTCA 601 (SEQ ID NO: 78)Bartonella X89208 TTGGGTGGTTTAC ACTGAGCAGT- hens. nt 761- TGCTCAGTAAACCACCCAA 781 (SEQ ID NO: 79) H. influenza M35019 AATTGAAGAGTTCATGATCAAA- nt 2-21 TGATCATG CTCTTCAATTN (SEQ ID NO: 80) S. thyphi.U88545 nt2-21 H. influenza M35019 TATTATCGGAAG CACTTTCATC- nt 180-ATGAAAGTG TTCCGATAATA 200 (SEQ ID NO: 81) H. influenza M35019AACTAGAGTACT CCTCCCTAAA- nt 649- TTAGGGAGG GTACTCTAGTT 669 (SEQ ID NO:82) H. influenza M35019 GGGGGTTGGGGT CAGAGTTAAA- nt 829- TTAACTCTGCCCCAACCCCC 849 (SEQ ID NO: 83) Shigella dys. X96966 TGGCTCAGATTGGCCAGCGTTC- nt 1-21 AACGCTGGC AATCTGAGCCA (SEQ ID NO: 84) E. coli X80725nt 11-31 S. thyphi. X96966 nt 20-40 N. X07714 gonorrhoea nt 21-41 H.influenza M35019 nt 20-40 Shigella dys. X96966 ACGTCGCAAGAC CCCTCTTTGG-nt 162- CAAAGAGGG TCTTGCGACGT 182 (SEQ ID NO: 85) S. thyphi. X96966 nt181- 201 Shigella dys. X96966 TGAGTCTCGTAG TACCCCCCTC- nt 633- AGGGGGGTATACGAGACTCA 653 (SEQ ID NO: 86) E. coli X80725 nt 644- 664 S. thyphi.X96966 nt 652- 672 Shigella dys. X96966 GTTGTGCCCTTGA GCCACGCCTC- nt813- GGCGTGGC AAGGGCACAAC 833 (SEQ ID NO: 87) E. coli X80725 nt 824- 844S. thyphi. X96966 nt 832- 852 Shigella dys. X96966 GAACCTTGTAGACCTCGTATCT- nt 983- GATAGGAGG CTACAAGGTTC 1003 (SEQ ID NO: 88) 3Approximate nucleotide locations

Exemplary Gram-positive bacterial targets include, but are not limitedto, Staphylococcus aureus, Mycobacterium tuberculosis and Streptococcuspneumoniae.

Exemplary oligomer sequences antisense to Gram-positive bacterial 16SrRNA sequences are exemplified in Table 3 by the sequences presented asSEQ ID NO:27 and SEQ ID NO:28, with the bacterial 16s rRNAs to which theexemplary antisense oligomers are targeted indicated in Table 3 as “+”and those which are not targeted indicated as “−”.

TABLE 3 GRAM POSITIVE 16s rRNA SEQUENCES AND ANTISENSE OLIGOMERSAACTACG- TCGTGAG- TGCCAGC ATGTTGG SEQUENCE AGCCGCG GTTAAGT CGCGGCT-ACTTAA- GCTGGCA CCCAACATC Organism ANTISENSE CGTAGTT TCACGA Staph aureusY15856 + + Myco. tubercul. X52917 + + Strep. pneumoniae AF003930 + + E.coli X80725 − − S. thyphi U88545 − − P. aeruginosa AF170358 − + Vibriocholera AF118021 − − N. gonorrhoea X07714 + + H. pylori M88157 − +Treponema pallad. AJ010951 − − Chlamydia trach. D85722 − − Bartonellahens X89208 − + H. influenza M35019 − − Shigella dys. X96966 − − 4 Basedon nucleotides 497-517 of GenBank Y15856, designated SEQ ID NO: 27 5Based on nucleotides 1064-1084 of GenBank Y15856, designated SEQ ID NO:28

Exemplary Gram-negative bacterial targets include, but are not limitedto, E. coli, Salmonella thyphimurium, Pseudomonas aeruginosa, Vibriocholera, Neisseria gonorrhoea, Helicobacter pylori, Bartonella henselae,Hemophilis Influenza and Shigella dysenterae.

Exemplary oligomer sequences antisense to Gram-negative bacterial 16SrRNA sequences are exemplified in Table 4 by the sequences presented asSEQ ID NO:29 and SEQ ID NO:30, with the bacterial 16s rRNAs to which theexemplary antisense oligomers are targeted indicated in Table 4 as “+”and those which are not targeted indicated as “−”.

TABLE 4 GRAM NEGATIVE 16s rRNA SEQUENCES AND ANTISENSE OLIGOMERSTCGGAAT- CCGCCCG- TACTGGGC TCACACCAT SEQUENCE GTAAA GGGAGT TTTACGC-ACTCCCA- CCAGTAATT TGGTGTGACG Organism ANTISENSE CCGA GGCGG E. coliX80725 + + S. thyphi U88545 + + P. aeruginosa AF170358 + + Vibriocholera AF118021 + + N. gonorrhoea X07714 + + Staph aureus Y15856 − −Myco. tubercul. X52917 − − H. pylori M88157 − + Strep. pneumoniaeAF003930 − − Treponema pallad. AJ010951 − + Chlamydia trach. D85722 − +Bartonella hens X89208 − + H. influenza M35019 − + Shigella dys.X96966 + + 6 Based on nucleotides 546-566 of GenBank X80725, designatedSEQ ID NO: 29 7 Based on nucleotides 1389-1409 of GenBank X80725,designated SEQ ID NO: 30

Exemplary bacterial targets for broad spectrum antisense oligomersinclude, but are not limited to, E. coli, Salmonella thyphimurium,Pseudomonas aeruginosa, Vibrio cholera, Neisseria gonorrhoea,Helicobacterpylori, Bartonella henselae, Hemophilis Influenza, Shigelladysenterae, Staphylococcus aureus, Mycobacterium tuberculosis,Streptococcus pneumoniae, Treponema palladium and Chlamydia trachomatis.(See Table 1.)

Exemplary broad spectrum antisense oligomers are presented in Tables 5Aand 5B as SEQ ID NOS:21-25, with the bacterial 16s rRNAs to which theexemplary antisense oligomers are targeted indicated in Tables 5A and 5Bas “+” and those which are not targeted indicated as “−”.

TABLE 5A BROAD SPECTRUM ANTISENSE OLIGONUCLEOTIDE SEQUENCES AGACTC-CGTGCC- AACAGG- CTACGG AGCAGC GATTAG SEQUENCE GAGGCAGCA CGCGGTAATATACCCTGGT TGCTGC- ATTACC- ACCAGG- CTCCCGT GCGGCT GTATC OrganismANTISENSE AGGAGTCT GCTGGCACG TAATCCTGTT E. coli X80725 + + + S. thyphiU88545 + + + P. aeruginosa AF170358 + + + Vibrio cholera AF118021 + + +N. gonorrhoea X07714 − + + Staph. aureus Y15856 + + + Myco. tubercul.X52917 + + + H. pylori M88157 + + + Strep. pneumoniae AF003930 + + +Treponema pallad. AJ010951 + + + Chlamydia trach. D85722 + + +Bartonella hens X89208 + + + H. influenza M35019 + + + Shigella dys.X96966 + + + 8: based on nucleotides 327-347 of GenBank No. X80725,designated SEQ ID NO: 21 9: based on nucleotides 504-524 of GenBank No.X80725, designated SEQ ID NO: 22 10: based on nucleotides 781-801 ofGenBank No. X80725, designated SEQ ID NO: 23

TABLE 5B BROAD SPECTRUM ANTISENSE OLIGONUCLEOTIDE SEQUENCES GCACAAG-ATGTTG- CGGTGGA GGTTAAGT SEQUENCE GCATGTG CCCGCAA CACATGC- TTGCGG-TCCACCG GACTTAAC Organism ANTISENSE CTTGTGC CCAACAT E. coli X80725 + +S. thyphi U88545 + + P. aeruginosa AF170358 + − Vibrio choleraAF118021 + + N. gonorrhoea X07714 − + Staph. aureus Y15856 + + Myco.tubercul. X52917 − + H. pylori M88157 − + Strep. pneumoniae AF003930 + +Treponema pallad. AJ010951 + − Chlamydia trach. D85722 − − Bartonellahens X89208 + + H. influenza M35019 + + Shigella dys. X96966 + + 11:based on nucleotides 924-944 of GenBank No. X80725, designated SEQ IDNO: 24 12: based on nucleotides 1072-1092 of GenBank No. X80725,designated SEQ ID NO: 25.

VI. Inhibitory Activity of Antisense Oligomers A. Effect of AntisenseOligomers to Bacterial 16S rRNA on Bacterial Growth

The effect of PMO antisense oligomers on bacterial culture viability wastested using the protocol described below; see “Bacterial Cultures” inMaterials and Methods. Briefly, test oligonucleotides, diluted inphosphate buffered saline (PBS), are added to the freshly inoculatedbacterial cultures; the cultures are incubated at 37° C. overnight,e.g., 6 to 26 hours, diluted, and plated on agar plates; colonies arecounted 16-24 hours later. Non-selective bacterial growth media, e.g.,agar containing nutrients appropriate to the type of bacteria beingcultured, are utilized, as generally known in the art.

The viability of bacteria following overnight culture with a testoligomer is based on the number of bacterial colonies in antisenseoligomer-treated cultures relative to untreated or nonsense treatedcultures. An exemplary nonsense control is an oligomer antisense toc-myc, having the sequence presented as SEQ ID NO:139.

A1. Inhibition of Salmonella thyphimurium with a Conserved-SequenceOligomer Antisense to 16S rRNA. Two strains of Salmonella thyphimurium(1535 and 1538) were inoculated into broth media, as described inMaterials and Methods, below. An oligomer antisense to a 16S rRNAsequence conserved amongst E. coli, S. thyphimurium and S. dysenterae(“BS-1”; SEQ ID NO:15) was added to a final concentration of 1 μM andthe tube placed in an incubator at 37° C. for 6 to 16 hours. At the endof the incubation, the broth was spread onto plates, incubated overnightfor 16 to 24 hours and colonies counted. The data, shown in Table 6,provides evidence that Salmonella thyphimurium is inhibited by a 16SrRNA antisense oligomer based on a 16S rRNA sequence which is conservedamongst E. coli, S. thyphimurium and S. dysenterae.

TABLE 6 Effect of Broad Spectrum Antisense on Salmonella thyphimuriumStrain Control 1 μM AS to 16S rRNA (culture time) (colonies) (colonies)% Inhibition 1535 (6 hours) 217 141 35 1535 (16 hours) 214  52 76 1538(6 hours) 824 664 19 1538 (16 hours) 670 133 80

A2. Effect of Antisense Oligomers to Bacterial 16S rRNA on Growth Of E.coli.

The effect of PMO antisense oligomers on inhibition of E. coli wasevaluated, using a procedure such as described above, by adding anantisense oligomer targeting particular 20-22 nucleotide portions of theE. coli 16S rRNA sequence found at GenBank Accession No. X80725 toindividual E. coli cultures. Each antisense oligomer was incubated at a1 μM concentration with E. coli bacteria for 16 hours, the cultures werediluted and plated on agar plates, and colonies were counted 16-24 hourslater. The results, shown in Table 7, indicate that PMO antisenseoligomers targeting E. coli 16S rRNA inhibited growth of colonies by upto 60%, with oligomers targeting various regions throughout the 16S rRNAsequence observed to be effective.

TABLE 7 E. coli 16s rRNA Targeting Study AVI SEQ Re- Ref. Loc- Antisensesequence ID Percent peats No. ation (5′→3′) NO. Inhibition S.E. (n)  91263- GCA CTT TAT GAG 19 59.8 3.4 8 1283 GTC CGC TTG 15 1272- GGA CTACGA CGC 15 19.5 7.4 9 1293 ACT TTA TGA G 16 1252- GGT CCG CTT GCT 1621.5 11 9 1272 CTC GCG AGG 17 446- GCA AAG GTA TTA 17 66 3.3 14 466 ACTTTA CTC 27  1- ATC TGA GCC ATG 97 55.2 9.7 5 20 ATC AAA CT 28 301- TGTCTC AGT TCC 98 35 7.2 8 320 AGT GTT GC 29 722- GTC TTC GTC CAG 99 52.5 47 741 GGG GCC GC 30 1021- CAC CTG TCT CAC 100 56 8.4 5 1040 GGT TCC CG31 1431- CGC CCT CCC GAA 101 43 13 5 1450 GTT AAG CT

FIG. 5 depicts the results of a study on the effect of variousconcentrations of the PMO having SEQ ID NO:15 (broad spectrum) targetedagainst a bacterial 16S rRNA consensus sequence on the bacterial colonyformation in E. coli, presented as percent inhibition of colonyformation. As the figure shows, about 70% inhibition was achieved atabout 0.1 μM PMO.

A3. Inhibition of Staphylococcus aureus and Pseudomonas aeruqinosa withOligomers Antisense to 16S rRNA.

Tables 8 and 9 show the effect of oligomers targeting 16S rRNA, at aconcentration of 1 μM, on bacterial growth in Staphylococcus aureus andPseudomonas aeruginosa. In a typical experiment, antisense oligomerstargeting particular 22-nucleotide portions of the Staphylococcus aureusand Pseudomonas aeruginosa 16S rRNA sequences, found at GenBankAccession Nos. Y15857 and Z76651, respectively, were incubated with therespective bacteria at a concentration of 1 μM for 16 hours. Growth ofS. aureus was inhibited by up to 25%, and growth of P. aeruginosa wasinhibited by up to about 53%.

TABLE 8 Staphylococcus aureus 16s rRNA Targeting Study AVI SEQ Ref.Antisense sequence ID Percent No. Location (5′→3′) NO Inhibition S.E. n= 23 447-466 ATG TGC ACA GTT 93 2.5 8.6 2 ACT TAC AC 24 1272- CTG AGAACA ACT 94 25.3 11 2 1291 TTA TGG GA

TABLE 9 Pseudomonas aeruginosa 16s rRNA Targeting Study AVI Antisensesequence SEQ ID Percent Ref. No. Location (5′→3′) NO: Inhibition S.E. n= 25 447-466 TTA TTC TGT TGG 95 37.3 9.8 3 TAA CGT CA 26 1272-1291 CGAGT TGC AGA CTG 96 52.7 7.1 3 CGA TC

Inhibition of Listeria was also demonstrated by a corresponding anti-16SPMO. A very low dose (about 30 nM) of the PMO gave about 40% inhibition.

A4. Effect of Antisense Oligomers to Bacterial rRNA on Growth OfVancomycin-Resistant Enterococcus feacium (VRE)

(a) Bacterial 16S rRNA Targets

The effect of PMO antisense oligomers on the growth of VRE wasevaluated, using the method described above, by adding antisense PMO'stargeting numerous 16S rRNA sequences to cultures of VRE and incubatingat a concentration of 1 μM for 16 hours. The results shown in Table 10and in FIG. 6 indicate that inhibition ranged from about 48% to about70%, averaging about 60%, with no significant differences ineffectiveness seen among the oligomers tested. (The nucleotide symbol“M” in the sequences represents methyl cytidine.)

FIG. 6 illustrates the effect of a broad spectrum PMO on VRE colonyformation. The oligomer designated SEQ ID NO:114 is considered broadspectrum, targeted to a region conserved in all of the bacteria listedin Table 5A, above. This oligomer targets approximately the same regionas that targeted by SEQ ID NO:23, which is shown in Table 5A. As can beseen from the data in Table 10, this oligomer was similar ineffectiveness to a “narrow spectrum” oligomer specific to Enterococcus,SEQ ID NO:115.

Also included were several oligomers specific to 16s rRNA of otherorganisms (E. coli, S. aureus, and P. aeruginosa). These oligomers hadno inhibitory effect on VRE.

TABLE 10 Targeting Study in Enterococcus faecium. PMO GenBank SEQPercent Source Acc. No. Location Antisense Sequence (5′→3′) IDInhibition S.E. n = VRE Y18294 447-466 GAT GAA CAG TTA CTC TCA TC 9161.7 2.7 3 VRE Y18294 1272-1291 ACT GAG AGA AGC TTT AAG AG 92 59.7 5.1 6VRE Y18294  1-20 GGC ACG CCG CCA GCG TTC G 102 56.7 7.8 3 VRE Y18294300-319 TGT CTC AGT CCC AAT GTG GC 103 53.7 1.0 3 VRE Y18294 721-740 GTTACA GAC CAG AGA GCC GC 104 69.7 3.0 3 VRE Y18294 1022-1041 CAC CTG TCACTT TGC CCC CG 105 47.9 10.1 3 VRE Y18294 1438-1456 GGC GGC TGG CTC CAAAAG G 106 58.5 3.2 3 VRE Y18294 776-795 GAC TAC CAG GGT ATC TAA TC 11462.2 5.5 3 VRE Y18294 194-213 CAG CGA CAC CCG AAA GCC CC 115 70.1 3.3 3S. aureus See Table 8 CTG AGA ACA ACT TTA TGG GA 94 24 8.8 3 P.aeruginosa See Table 9 TCG AGT TGC AGA CTG CGA TC 96 26 11.6 3 E. coliSee Table 7 GCA AAG GTA TTA ACT TTA 17 17 22.4 3 CTC E. coli See Table 7GCA CTT TAT GAG GTC CGC 19 9 10 3 TTG VRE Y18294 0077-95  CAC CCG TTCGCC ACT CCT C 107 45.1 6.1 3 VRE Y18294 0895-914  TCA ATT CCT TTG AGTTTC AA 108 31.8 15.3 3 VRE Y18294 1263-1291 GCA ATC CGC ACT GAG AGA 10939.1 11.4 6 AGC TTT AAG AG VRE Y18294 1268-1291 C CGC ACT GAG AGA AGCTTT 110 50.1 5.5 6 AAG AG VRE Y18294 1275-1291 GAG AGA AGC TTT AAG AG111 61.5 3.3 6 VRE Y18294 1277-1291 G AGA AGC TTT AAG AG 112 46.3 5 6VRE Y18294 1282-1291 A AGC TTT AAG AG 113 39.5 8.2 6 VRE Y182941274-1291 T GAG AGA AGC TTT AAG AG 121 57.2 4.8 3 VRE Y18294 1273-1291CT GAG AGA AGC TTT AAG AG 122 54.4 2.7 3 VRE Y18294 196-213 GCG ACA CCCGAA AGC GCC 123 59.0 5.3 6 VRE Y18294 723-740 TAC AGA CCA GAG AGC CGC124 63.3 4.9 9 VRE Y18294 197-213 CGA CAC CCG AAA GCG CC 125 63.6 3.7 9VRE Y18294 195-213 A GCG ACA CCC GAA AGC GCC 126 60.6 4.8 12  VRE Y18294196-213 CG ACA CCC GAA AGC GCC A 127 58.9 5.6 9 VRE Y18294 197-213 MGAMA MMM GAA AGM GMM 128 60.3 4.5 9 VRE Y18294 723-740 TAM AGA MMA GAGAGM MGM 129 56.9 3.9 9 VRE Y18294 1162-1177 MMM MAM MTT MTT MMG G 13056.1 3.7 9 VRE Y18294 1345-1363 CAC CGC GGC GTG CTG ATC C 131 64.0 3.9 6VRE Y18294 1162-1177 CCC CAC CTT CCT CCG G 132 70.2 1.6 3 VRE Y18294916-933 CCG CTT GTG CGG GCC CCC 133 66.8 4.3 3 VRE Y18294 1345-1362 CACCGC GGC GTG CTG ATC 134 71.4 11.3 3 VRE Y18294 1345-1361 CAC CGC GGC GTGCTG AT 135 57.3 3.8 3 VRE Y18294 1346-1364 ACC GCG GCG TGC TGA TCC 13675.0 4.4 3 VRE Y18294 1344-1360 CCG CGG CGT GCT GAT CC 137 66.3 3.5 3VRE Y18294 1346-1363 ACC GCG GCG TGC TGA TC 138 63.8 2.2 3 M representsmethyl cytidine.

A dose-response study was also conducted using different concentrationsof the oligomer having SEQ ID NO:92. About 70% inhibition was achievedat 1-10 μM, about 50% at 0.1 μM, about 20% at 0.01 μM, and about 12% at1 nM.

(b) Bacterial 23S rRNA Targets

In a related experiment, also using vancomycin-resistant Enterococcusfeacium (VRE) as the target bacteria, the effect of PMO antisenseoligomers targeting 23S rRNA sequences on bacterial growth wasevaluated, using the method described above. In individual assays,antisense PMO's targeting VRE 23S rRNA sequences were added to culturesof VRE and incubated at a concentration of 1 μM for 16 hours. The datain Table 11, below, represented graphically in FIG. 7, shows thatantisense targeting of 23S rRNA in VRE was successful in inhibitingbacterial growth. Locations refer to GenBank Acc. No. X79341.

TABLE 11 VRE 23S rRNA Targeting Study Ref. SEQ ID Percent S.E. No.Location Antisense Sequence (5′→3′) NO: Inhibition (N = 3) 46 20-39 GTGCCA AGG CAT CCA CCG TG 116 61.9 4.6 47 679-698 CAT ACT CAA ACG CCC TATTC 117 46.8 6.6 48 1462-1480 CCT TAG CCT CCT GCG TCC C 118 47.6 7.5 492060-2079 GGG GTC TTT CCG TCC TGT CG 119 67.0 5.7 50 2881-2900 CGA TCGATT AGT ATC AGT CC 120 63.0 10.5 

B. Effect of Length of Antisense Oligomer on Inhibition of VRE

The procedure used to obtain the data shown in Table 10, above, wasrepeated using different-length versions (SEQ ID NOS:109-113) of theanti-16S rRNA oligomer having SEQ ID No:92, ranging from a 12-mer (SEQID NO:113) to a 29-mer (SEQ ID NO:109). Results are given in Table 12,below.

As shown in Table 12 and FIG. 8, the optimum length in this study was inthe 17- to 20-mer range. Further studies confirmed that oligomers with alength of from 17 to 20 nucleotide subunits, and more preferably 17-18subunits, are generally preferred. The results suggest that shorteroligomers, such as 12-mers, may have insufficient binding affinity, andthat longer oligomers, such as the 29-mer, are less easily transportedinto cells.

TABLE 12 Antisense Targeting of 16S rRNA in VRE Ref. SEQ ID Percent No.length Antisense sequence (5′→3′) NO: Inhibition SE n = 39 29 mer GCAATC CGC ACT GAG AGA AGC 109 29.1 11.4  6 TTT AAG AG 40 24 mer C CGC ACTGAG AGA AGC TTT 110 51.1 5.5 6 AAG AG 22 20 mer ACT GAG AGA AGC TTT AAGAG  92 59.7 5.2 6 41 17 mer GAG AGA AGC TTT AAG AG 111 61.5 3.3 6 42 15mer G AGA AGC TTT AAG AG 112 46.3 5.0 6 43 12 mer A AGC TTT AAG AG 11339.5 8.2 6

C. Antisense PMO Resistance Study in VRE

The 20-mer anti-16S rRNA antisense oligomer referred to above (SEQ IDNO:92) was used in a resistance study with VRE. After each day ofincubation (concn. 1 μM), three colonies were picked and retreated witholigomer to test for resistance. As shown in Table 13, below, and inFIG. 9, viability increased somewhat at four days but then dropped againat five and six days. Tests carried out to twelve days (data not shown)showed no evidence that resistance to the oligomer had developed.

TABLE 13 Resistance Study with anti-16S rRNA (SEQ ID NO: 92) in VRE DayPercent Inhibition S.E. (n = 3) 1 41.8  5.2 2 49.6  2.7 3 51.8 12.3 419.2 11.9 5 34.1 10.9 6 47.2 12.0

D. Combination Therapy with Antibiotic Drugs

Enterococcus faecium was treated with vancomycin alone and incombination with 1.0 μM antisense PMO targeted to VRE 16S rRNA (SEQ IDNO:92). Inhibition was greatly increased by addition of the PMO, asshown in FIG. 10A, and the organisms were completely eliminated at 3 μMvancomycin and 1 μM PMO. The results show that use of an antisense PMOtargeted to VRE 16S rRNA together with vancomycin results in an enhancedanti-bacterial effect relative that of vancomycin alone.

A similar study was conducted with vancomycin resistantEnterococcusfaecium (VRE), treated with ampicillin alone and incombination with 1.0 μM of the same antisense PMO (see FIG. 10B). Again,essentially complete inhibition was achieved by the combination at 3 μMampicillin. Similar to the results obtained for vancomycin, thecombination of an antisense PMO targeted to VRE 16S rRNA and ampicillinresulted in an enhanced anti-bacterial effect.

VII. In Vivo Administration Of Antisense Oliaomers

In another aspect, the invention is directed to slowing or limitingbacterial infection in vivo in a mammal, and/or decreasing oreliminating detectable symptoms typically associated with infection bythat particular bacteria. In general, a therapeutically effective amountof an antisense oligonucleotide-containing pharmaceutical composition isadministered to a mammalian subject, in a manner effective to inhibitthe activity of a 16S rRNA.

The antisense oligonucleotides of the invention and pharmaceuticalcompositions containing them are useful for inhibiting bacterialinfection in vivo in a mammal, and for inhibiting or arresting thegrowth of bacteria in the host. In other words, the bacteria may bedecreased in number or eliminated, with little or no detrimental effecton the normal growth or development of the host.

In some cases, the antisense oligomer will inhibit the growth ofbacteria in general. In other cases, the antisense oligomer will bespecific to one or more particular types of bacteria, e.g. a particulargenus, species or strain.

It will be understood that the in vivo efficacy of such an antisenseoligomer in a subject using the methods of the invention is dependentupon numerous factors including, but not limited to, (1) the targetsequence; (2) the duration, dose and frequency of antisenseadministration; and (3) the general condition of the subject.

The efficacy of an in vivo administered antisense oligomer of theinvention on inhibition or elimination of the growth of one or moretypes of bacteria may be determined by in vitro culture or microscopicexamination of a biological sample (tissue, blood, etc.) taken from asubject prior to, during and subsequent to administration of theantisense oligomer. (See, for example, Pari, G. S. et al., Antimicrob.Agents and Chemotherapy 39(5):1157-1161, 1995; Anderson, K P et al.,Antimicrob. Agents and Chemotherapy 40(9):2004-2011, 1996.)

A. Treating Subjects

Effective delivery of the antisense oligomer to the target RNA is animportant aspect of the methods of the invention. In accordance with theinvention, such routes of antisense oligomer delivery include, but arenot limited to, various systemic routes, including oral and parenteralroutes, e.g., intravenous, subcutaneous, intraperitoneal, andintramuscular, as well as inhalation, transdermal and topical delivery.The appropriate route may be determined by one of skill in the art, asappropriate to the condition of the subject under treatment.

For example, an appropriate route for delivery of an antisense oligomerin the treatment of a bacterial infection of the skin is topicaldelivery, while delivery of an antisense oligomer in the treatment of abacterial respiratory infection is by inhalation.

Additional exemplary embodiments include oral delivery of an antisenseoligomer directed to bacterial 16S or 23S rRNA for treatment of aurinary tract infection or sepsis and IV delivery for treatment ofsepsis.

It is appreciated that methods effective to deliver the oligomer to thesite of bacterial infection or to introduce the oligonucleotide into thebloodstream are contemplated.

Transdermal delivery of antisense oligomers may be accomplished by useof a pharmaceutically acceptable carrier adapted for topicaladministration. One example of morpholino oligomer delivery is describedin PCT patent application WO 97/40854, incorporated herein by reference.

In one aspect of the invention, an antisense oligomer directed tobacterial 16S or 23S rRNA is delivered by way of a catheter,microbubbles, a heart valve coated or impregnated with oligomer, aHickman catheter or a coated stent.

In one preferred embodiment, the oligomer is a morpholino oligomer,contained in a pharmaceutically acceptable carrier, and deliveredorally. In a further aspect of this embodiment, a morpholino antisenseoligonucleotide is administered at regular intervals for a short timeperiod, e.g., daily for two weeks or less. However, in some cases theantisense oligomer is administered intermittently over a longer periodof time.

Typically, one or more doses of antisense oligomer are administered,generally at regular intervals, for a period of about one to two weeks.Preferred doses for oral administration are from about 1 mgoligomer/patient to about 25 mg oligomer/patient (based on a weight of70 kg). In some cases, doses of greater than 25 mg oligomeripatient maybe necessary. For IV administration, the preferred doses are from about0.5 mg oligomer/patient to about 10 mg oligomer/patient (based on anadult weight of 70 kg).

The antisense compound is generally administered in an amount and mannereffective to result in a peak blood concentration of at least 200-400 nMantisense oligomer.

In general, the method comprises administering to a subject, in asuitable pharmaceutical carrier, an amount of an antisense agenteffective to inhibit the biological activity of a bacterial 16S or 23SrRNA target sequence of interest.

It follows that a morpholino antisense oligonucleotide composition maybe administered in any convenient vehicle which is physiologicallyacceptable. Such an oligonucleotide composition may include any of avariety of standard pharmaceutically accepted carriers employed by thoseof ordinary skill in the art. Examples of such pharmaceutical carriersinclude, but are not limited to, saline, phosphate buffered saline(PBS), water, aqueous ethanol, emulsions such as oil/water emulsions,triglyceride emulsions, wetting agents, tablets and capsules. It will beunderstood that the choice of suitable physiologically acceptablecarrier will vary dependent upon the chosen mode of administration.

In some instances liposomes may be employed to facilitate uptake of theantisense oligonucleotide into cells. (See, e.g., Williams, S. A.,Leukemia 10(12):1980-1989, 1996; Lappalainen et al., Antiviral Res.23:119, 1994; Uhlmann et al., ANTISENSE OLIGONUCLEOTIDES: A NEWTHERAPEUTIC PRINCIPLES, Chemical Reviews, Volume 90, No. 4, pages544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers inBiology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels mayalso be used as vehicles for antisense oligomer administration, forexample, as described in WO 93/01286. Alternatively, theoligonucleotides may be administered in microspheres or microparticles.(See, e.g., Wu GY and Wu CH, J. Biol. Chem. 262:4429-4432, 1987.)

Sustained release compositions are also contemplated within the scope ofthis application. These may include semipermeable polymeric matrices inthe form of shaped articles such as films or microcapsules.

In one aspect of the method, the subject is a human subject, typically asubject diagnosed as having a localized or systemic bacterial infection.

In another aspect, the condition of the patient may dictate prophylacticadministration of an antisense oligomer of the invention, i.e., apatient who (1) is immunocompromised; (2) is a burn victim; (3) has anindwelling catheter; (4) is about to undergo or has recently undergonesurgery, etc.

In another application of the method, the subject is a livestock animal,e.g., a chicken, turkey, pig, cow or goat, etc., and the treatment iseither prophylactic or therapeutic.

In addition, the methods of the invention are applicable to treatment ofany condition wherein inhibiting or eliminating the growth of bacteriawould be effective to result in an improved therapeutic outcome for thesubject under treatment.

It will be understood that an effective in vivo treatment regimen usingthe antisense oligonucleotides of the invention will vary according tothe frequency and route of administration, as well as the condition ofthe subject under treatment (i.e., prophylactic administration versusadministration in response to localized or systemic infection).Accordingly, such in vivo therapy will generally require monitoring bytests appropriate to the particular type of bacterial infection undertreatment and a corresponding adjustment in the dose or treatmentregimen in order to achieve an optimal therapeutic outcome.

B. Monitoring Treatment

The efficacy of a given therapeutic regimen involving the methodsdescribed herein may be monitored, e.g., by general indicators ofinfection, such as complete blood count (CBC), nucleic acid detectionmethods, immunodiagnostic tests or bacterial culture.

Identification and monitoring of bacterial infection generally involvesone or more of (1) nucleic acid detection methods; (2) serologicaldetection methods, i.e., conventional immunoassay; (3) culture methods;and (4) biochemical methods. Such methods may be qualitative orquantitative.

DNA probes may be designed based on publicly available bacterial nucleicacid sequences, and used to detect target genes or metabolites (i.e.,toxins) indicative of bacterial infection, which may be specific to aparticular bacterial type, e.g., a particular species or strain, orcommon to more than one species or type of bacteria (i.e., Gram positiveor Gram negative bacteria). In addition, nucleic amplification tests(e.g., PCR) may be used in such detection methods.

Serological identification may be accomplished using a bacterial sampleor culture isolated from a biological specimen, e.g., stool, urine,cerebrospinal fluid, blood, etc. Immunoassay for the detection ofbacteria is generally carried out by methods routinely employed by thoseof skill in the art, e.g., ELISA or Western blot.

In general, procedures and/or reagents for immunoassay of bacterialinfections are routinely employed by those of skill in the art. Inaddition, monoclonal antibodies specific to particular bacterial strainsor species are often commercially available.

Culture methods may be used to isolate and identify particular types ofbacteria, by employing techniques including, but not limited to, aerobicversus anaerobic culture, and growth and morphology under variousculture conditions.

Exemplary biochemical tests include Gram stain (Gram, 1884; Grampositive bacteria stain dark blue, and Gram negative stain red),enzymatic analyses (i.e., oxidase, catalase positive for Pseudomonasaeruginosa), and phage typing.

It will be understood that the exact nature of such diagnostic, andquantitative tests as well as other physiological factors indicative ofbacterial infection will vary dependent upon the bacterial target, thecondition being treated and whether the treatment is prophylactic ortherapeutic.

In cases where the subject has been diagnosed as having a particulartype of bacterial infection, the status of the bacterial infection isalso monitored using diagnostic techniques typically used by those ofskill in the art to monitor the particular type of bacterial infectionunder treatment.

The antisense oligomer treatment regimen may be adjusted (dose,frequency, route, etc.), as indicated, based on the results ofimmunoassays, other biochemical tests and physiological examination ofthe subject under treatment.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications may be made without departing from the invention.

MATERIALS AND METHODS

Standard recombinant DNA techniques were employed in all constructions,as described in Ausubel, FM, et al., in CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley and Sons, Inc., Media, Pa., 1992 and Sambrook J, etal., in MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., Vol. 2, 1989), both of whichare expressly incorporated by reference herein.

Plasmid. The plasmid used for studies in support of the presentinvention was engineered using pCi-Neo mammalian expression vector(Promega), by inserting 36 bases of the c-myc target region along withthe coding region for firefly luciferase into the vector in thepolylinker downstream from the T7 promoter. The A from the ATG of codonNo. 1 of luciferase was removed by in vitro mutagenesis, leaving the ATGthat is present in the c-myc sequence in frame with the reporter. Theplasmid, pCiNeo(myc)lucδA, also contained the b-lactamase gene codingfor antibiotic resistance and was transformed into Escherichia Coli DH5.

Bacterial Cultures. In evaluating the effectiveness of antisenseoligonucleotides of the invention, approximately 3 ml bacterial cultureswere aliquoted into plastic snap cap tubes from a 45 ml starting culturein Luria-Bertani (LB) Broth containing 4.5 mg of Ampicillin and a singlebacterial colony taken from a freshly streaked LB agar plate containing100 μg/mL ampicillin. The test oligomer diluted in phosphate bufferedsaline (PBS) was added to the cultures, incubated at 37° C. for aspecific time, e.g., 16 or 26 hours with shaking at 210 rpm, then placedon ice for 15 minutes.

Culture staining microscopy and colony scanning. Bacterial plate countsrequire that a measured volume of material be added to agar either bythe pour plate or spread plate technique. If the original sample has alarge number of bacteria, dilutions are prepared and plated. The platesare incubated, and the number of colony-forming units (CFU) reflect theviable organisms in the sample. The colonies may be counted manuallyusing a microscope; however, it is preferred that an automatic colonycounter be employed (e.g., as offered by Bioscience International,Rockville, Md.). Bacterial cultures are stained in accordance withstandard Gram staining protocols. The stained bacterium are visualizedusing a Nikon Optiphot-2 upright microscope, with images magnified 1000×using the combination of an 100× oil immersion lens and the 10×magnification of the camera. The camera used to capture the images is aNikon N8008S. The images are taken using bright field microscopy with a4 second exposure on a setting 5 light output. A preferred film wasKodak Gold 400 ASA. After developing, the images are scanned using aMicrotek Scan Maker 4, then cropped using Adobe PhotoShop.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 139 <210> SEQ ID NO 1 <211> LENGTH: 1450<212> TYPE: DNA <213> ORGANISM: Escherichia coli <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1450)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 1agtttgatca tggctcagat tgaacgctgg cggcaggcct aacacatgca ag#tcgaacgg      60taacaggaag cagcttgctg ctttgctgac gagtggcgga cgggtgagta at#gtctggga     120aactgcctga tggaggggga taactactgg aaacggtagc taataccgca ta#acgtcgca     180agcacaaaga gggggacctt agggcctctt gccatcggat gtgcccagat gg#gattagct     240agtaggtggg gtaacggctc acctaggcga cgatccctag ctggtctgag ag#gatgacca     300gcaacactgg aactgagaca cggtccagac tcctacggga ggcagcagtg gg#gaatattg     360cacaatgggc gcaagcctga tgcagccatg cngcgtgtat gaagaaggcc tt#cgggttgt     420aaagtacttt cagcggggag gaagggagta aagttaatac ctttgctcat tg#acgttacc     480cgcagaagaa gcaccggcta actccgtgcc agcagccgcg gtaatacgga gg#gtgcaagc     540gttaatcgga attactgggc gtaaagcgca cgcaggcggt ttgttaagtc ag#atgtgaaa     600tccccgggct caacctggga actgcatctg atactggcaa gcttgagtct cg#tagagggg     660ggtagaattc caggtgtagc ggtgaaatgc gtagagatct ggaggaatac cg#gtggcgaa     720ggcggccccc tggacgaaga ctgacgctca ggtgcgaaag cgtggggagc aa#acaggatt     780agataccctg gtagtccacg ccgtaaacga tgtcgacttg gaggttgtgc cc#ttgaggcg     840tggcttccgg anntaacgcg ttaagtcgac cgcctgggga gtacggccgc aa#ggttaaaa     900ctcaaatgaa ttgacggggg ccgcacaagc ggtggagcat gtggtttaat tc#gatgcaac     960gcgaagaacc ttacctggtc ttgacatcca cggaagtttt cagagatgag aa#tgtgcctt    1020cgggaaccgt gagacaggtg ctgcatggct gtcgtcagct cgtgttgtga aa#tgttgggt    1080taagtcccgc aacgagcgca acccttatcc tttgttgcca gcggtccggc cg#ggaactca    1140aaggagactg ccagtgataa actggaggaa ggtggggatg acgtcaagtc at#catggccc    1200ttacgaccag ggctacacac gtgctacaat ggcgcataca aagagaagcg ac#ctcgcgag    1260agcaagcgga cctcataaag tgcgtcgtag tccggattgg agtctgcaac tc#gactccat    1320gaagtcggaa tcgctagtaa tcgtggatca gaatgccacg gtgaatacgt tc#ccgggcct    1380tgtacacacc gcccgtcaca ccatgggagt gggttgcaaa agaagtaggt ag#cttaactt    1440 cgggagggcg                 #                  #                   #       1450 <210> SEQ ID NO 2 <211> LENGTH: 1541<212> TYPE: DNA <213> ORGANISM: Salmonella thyphimurium<400> SEQUENCE: 2aaattgaaga gtttgatcat ggctcagatt gaacgctggc ggcaggccta ac#acatgcaa      60gtcgaacggt aacaggaagc agcttgctct ttgctgacga gtggcggacg gg#tgagtaat     120gtctgggaaa ctgcctgatg gagggggata actactggaa acggtggcta at#accgcata     180acgtcgcaag accaaagagg gggaccttcg ggcctcttgc catcggatgt gc#ccagatgg     240gattagctag taggtggggt aacggctcac ctaggcgacg atccctagct gg#tctgagag     300gatgaccagc cacactgaag ctgaagcacg gtccagactc ctacgggagg ca#gcagtggg     360gaatattgca caatgggcgc aagcctgatg cagccatgcc gcgtgtatga ag#aaggcctt     420cgggttgtaa agtactttca gcggggagga aggtgttgtg gttaataacc gc#agcaattg     480acgttacccg cagaagaagc accggctaac tccgtgccag cagccgcggt aa#tacggagg     540gtgcaagcgt taatcggaat tactgggcgt aaagcgcacg caggcggttt gt#taagtcag     600atgtgaaatc cccgggctca acctgggaac tgcatctgat actggcaagc tt#gagtctcg     660tagagggggg tagaattcca ggtgtagcgg tgaaatgcgt agagatctgg ag#gaataccg     720gtggcgaagg cggccccctg gacgaagact gacgctcagg tgcgaaagcg tg#gggagcaa     780acaggattag ataccctggt agtccacgcc gtaaacgatc tctacttgga gg#ttgtgccc     840ttgaggcgtg gcttccggag ctaacgcgtt aagtagagtg cttggggagt ac#ggccgcaa     900ggttaaaact caaatgaatt gacgggggcc cgcacaagcg gtggagcatg tg#gtttaatt     960cgatgcaacg cgaagaacct tacctggtct tgacatccac agaactttcc ag#agatgaga    1020ttgtgccttc gggaactgtg agacaggtgc tgcatggctg tcgtcagctc gt#gttgtgaa    1080atgttgggtt aagtcccgca acgagcgcaa cccttatcct ttgttgccag cg#gtccggcc    1140gggaactcaa aggagactgc cagtgataaa ctggaggaag gtggggatga cg#tcaagtca    1200tcatggccct tacgaccagg gctacacacg tgctacaatg gcgcatacaa ag#agaagcga    1260cctcgcgaga gcaagcggac ctcataaagt gcgtcgtagt ccggattgga gt#ctgcaact    1320cgactccatg aagtcggaat cgctagtaat cgtggatcag aatgccacgg tg#aatacgtt    1380cccgggcctt gtacacaccg cccgtcacac catgggagtg ggttgcaaaa ga#agtaggta    1440gcttaacctt cgggagggcg cttaccactt tgtgattcat gactggggtg aa#gtcgtaac    1500 aaggtaaccg taggggaacc tgcggttgga tcacctcctt a    #                   #  1541 <210> SEQ ID NO 3 <211> LENGTH: 1467<212> TYPE: DNA <213> ORGANISM: Pseudomonas aeruginosa <400> SEQUENCE: 3atgaagaggg cttgctctct gattcagcgg cggacgggtg agtaatgcct ag#gaatctgc      60ctgatagtgg gggacaacgt ttcgaaagga acgctaatac cgcatacgtc ct#acgggaga     120aagcagggga ccttcgggcc ttgcgctatc agatgagcct aggtcggatt ag#ctagttgg     180tgaggtaacg gctcaccaag gcgacgatcc gtaactggtc tgagaggatg at#cagtcaca     240ctggaactga gacacggtcc agactcctac gggaggcagc agtggggaat at#tggacaat     300gggcgaaagc ctgatccagc catgccgcgt gtgtgaagaa ggtcttcgga tt#gtaaagca     360ctttaagttg ggaggaaggg cattaaccta atacgttagt gttttgacgt ta#ccgacaga     420ataagcaccg gctaacttcg tgccagcagc cgcggtaata cgaagggtgc aa#gcgttaat     480cggaattact gggcgtaaag cgcgcgtagg tggtttgtta agttgaatgt ga#aagccccg     540ggctcaacct gggaactgca tccaaaactg gcaagctaga gtatggcaga gg#gtggtgga     600atttcctgtg tagcggtgaa atgcgtagat ataggaagga acaccagtgg cg#aaggcgac     660cacctgggct aatactgaca ctgaggtgcg aaagcgtggg gagcaaacag ga#ttagatac     720cctggtagtc cacgccgtaa acgatgtcga ctagccgttg ggatccttga ga#tcttagtg     780gcgcagctaa cgcattaagt cgaccgcctg gggagtacgg ccgctaggtt aa#aactctaa     840tgaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gc#aacgcgaa     900gaaccttacc aggccttgac atgcagagaa ctttccagag atggattggt gc#cttcggga     960actctgacac aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt tg#ggttaagt    1020cccgtaacga gcgcaaccct tgtccttagt taccagcacg ttaaggtggg ca#ctctaagg    1080agactgccgg tgacaaaccg gaggaaggtg gggatgacgt caagtcatca tg#gcccttac    1140ggcctgggct acacacgtgc tacaatggtc ggtacaaagg gttgccaagc cg#cgaggtgg    1200agctaatccc ataaaaccga tcgtagtccg gatcgcagtc tgcaactcga ct#gcgtgaag    1260tcggaatcgc tagtaatcgt gaatcagaat gtcacggtga atacgttccc gg#gccttgta    1320cacaccgccc gtcacaccat gggagtgggt tgctccagaa gtagctagtc ta#accttcgg    1380ggggacggtt accacggagg tattcatgac tggggtgaag tcgtaacaag gt#agccgtag    1440 gggaacctgc ggctggatca cctcctt          #                   #            1467 <210> SEQ ID NO 4<211> LENGTH: 1500 <212> TYPE: DNA <213> ORGANISM: Vibrio cholera<400> SEQUENCE: 4attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggtaacattt ca#aaagcttg      60cttttgaaga tgacgagcgg cggacgggtg agtaatggct gggaacctgc cc#tgacgtgg     120gggataacag ttggaaacga ctgctaatac cgcatgatgt ttacggacca aa#gaggggga     180tyttcggacy tytcgcgtcg ggatgggccc agttgggatt agctagttgg tg#aggtaatg     240gctcaccaag gcgacgatcc ctagctggtt tgagaggatg atcagccaca ct#ggaactga     300gacacggtcc agactcctac gggaggcagc agtggggaat attgcacaat gg#gcgcaagc     360ctgatgcagc catgccgcgt gtgtgaagaa ggccttcggg ttgtaaagca ct#ttcagcag     420tgaggaaggt tggtgcgtta atagcgtatc aatttgacgt tagctgcaga ag#aagcaccg     480gctaactccg tgccagcagc cgcggtaata cggagggtgc gagcgttaat cg#gaattact     540gggcgtaaag cgcatgcagg cggtttgtta agcaagatgt gaaagccccg gg#ctcaacct     600gggaaccgca ttttgaactg gcaggctaga gtcttgtaga ggggggtaga at#ttcaggtg     660tagcggtgaa atgcgtagag atctgaagga ataccggtgg cgaaggcggc cc#cctggaca     720aagactgacg ctcagatgcg aaagcgtggg gagcaaacag gattagatac cc#tggtagtc     780cacgctgtaa acgatgtcta cttggaggtt gtgacctara gtcgtggctt tc#ggagctaa     840cgcgttaagt agaccgcctg gggagtacgg tcgcaagatt aaaactcaaa tg#aattgacg     900ggggcccgca caagcggtgg agcatgtggt ttaattcgat gcaacgcgaa ga#accttacc     960tactcttgac atccagagaa gccgaaagag attttggtgt gccttcggga ac#tctgagac    1020aggtgctgca tggctgtcgt cagctcgtgt tgtgaaatgt tgggttaagt cc#cgcaacga    1080gcgcaaccct tatccttgtt tgccagcgag taatgtcggg aactccaggg ag#actgccgg    1140tgataaaccg gaggaaggtg gggacgacgt caagtcatca tggcccttac ga#gtagggct    1200acacacgtgc tacaatggca tatacagagg gcagcgaggc cgcgaggtgg ag#cgaatccc    1260agaaagtatg tcgtagtccg gatcggagtc tgcaactcga ctccgtgaag tc#ggaatcgc    1320tagtaatcgt gaatcagaat gtcacggtga atacgttccc gggccttgta ca#caccgccc    1380gtcacaccat gggagtgggc tgcaccagaa gtagatagct taaccttcgg ga#gggcgttt    1440accacggtgt ggttcatgac tggggtgaag tcgtaacaag gtagccctag gg#gaacctgg    1500 <210> SEQ ID NO 5 <211> LENGTH: 1544 <212> TYPE: DNA<213> ORGANISM: Neisseria gonorrhoea <400> SEQUENCE: 5tgaacataag agtttgatcc tggctcagat tgaacgctgg cggcatgctt ta#cacatgca      60agtcggacgg cagcacaggg aagcttgctt ctcgggtggc gagtggcgaa cg#ggtgagta     120acatatcgga acgtaccggg tagcggggga taactgatcg aaagatcagc ta#ataccgca     180tacgtcttga gagggaaagc aggggacctt cgggccttgc gctatccgag cg#gccgatat     240ctgattagct ggttggcggg gtaaaggccc accaaggcga cgatcagtag cg#ggtctgag     300aggatgatcc gccacactgg gactgagaca cggcccagac tcctacggga gg#cagcagtg     360gggaattttg gacaatgggc gcaagcctga tccagccatg ccgcgtgtct ga#agaaggcc     420ttcgggttgt aaaggacttt tgtcagggaa gaaaaggctg ttgccaatat cg#gcggccga     480tgacggtacc tgaagaataa gcaccggcta actacgtgcc agcagccgcg gt#aatacgta     540gggtgcgagc gttaatcgga attactgggc gtaaagcggg cgcagacggt ta#cttaagca     600ggatgtgaaa tccccgggct caacccggga actgcgttct gaactgggtg ac#tcgagtgt     660gtcagaggga ggtggaattc cacgtgtagc agtgaaatgc gtagagatgt gg#aggaatac     720cgatggcgaa ggcagcctcc tgggataaca ctgacgttca tgtccgaaag cg#tgggtagc     780aaacaggatt agataccctg gtagtccacg ccctaaacga tgtcaattag ct#gttgggca     840acttgattgc ttggtagcgt agctaacgcg tgaaattgac cgcctgggga gt#acggtcgc     900aagattaaaa ctcaaaggaa ttgacgggga cccgcacaag cggtggatga tg#tggattaa     960ttcgatgcaa cgcgaagaac cttacctggt tttgacatgt gcggaatcct cc#ggagacgg    1020aggagtgcct tcgggagccg taacacaggt gctgcatggc tgtcgtcagc tc#gtgtcgtg    1080agatgttggg ttaagtcccg caacgagcgc aacccttgtc attagttgcc at#cattcggt    1140tgggcactct aatgagactg ccggtgacaa gccggaggaa ggtggggatg ac#gtcaagtc    1200ctcatggccc ttatgaccag ggcttcacac gtcatacaat ggtcggtaca ga#gggtagcc    1260aagccgcgag gcggagccaa tctcacaaaa ccgatcgtag tccggattgc ac#tctgcaac    1320tcgagtgcat gaagtcggaa tcgctagtaa tcgcaggtca gcatactgcg gt#gaatacgt    1380tcccgggtct tgtacacacc gcccgtcaca ccatgggagt gggggatacc ag#aagtaggt    1440agggtaaccg caaggagtcc gcttaccacg gtatgcttca tgactggggt ga#agtcgtaa    1500 caaggtagcc gtaggggaac ctgcggctgg atcacctcct ttct   #                   #1544 <210> SEQ ID NO 6 <211> LENGTH: 1484<212> TYPE: DNA <213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 6ctggctcagg atgaacgctg gcggcgtgcc taatacatgc aagtcgagcg aa#cggacgag      60aagcttgctt ctctgatgtt agcggcggac gggtgagtaa cacgtggata ac#ctacctat     120aagactggga taacttcggg aaaccggagc taataccaga taatattttg aa#ccgcatgg     180ttcaaaagtg aaagacggtc ttgctgtcac ttatagatgg atccgcgctg ca#ttagctag     240ttggtaaggt aacggcttac caaggcaacg atgcatagcc gacctgagag gg#tgatcgkc     300cacactggaa ctgagacacg gtccagactc ctacgggagg cagcagtagg ga#atcttccg     360caatgggcga aagcctgacg gagcaacgcc gcgtgagtga tgaaggtctt cg#gatcgtaa     420aactctgtta ttagggaaga acatatgtgt aagtaactgt gcacatcttg ac#ggtaccta     480atcagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg tg#gcaagcgt     540tatccggaat tattgggcgt aaagcgcgcg taggcggttt ttyaagtctg at#gtgaaagc     600ccacggctca accgtggagg gtcattggaa actggaaaac ttgagtgcag aa#gaggaaag     660tggaattcca tgtgtagcgg tgaaatgcgc agagatatgg aggaacacca gt#ggcgaagg     720cgactttctg gtctgtaact gacgctgatg tgcgaaagcg tggggatcaa ac#aggattag     780ataccctggt agtccacgcc gtaaacgatg agtgctargt gttagggggt tt#ccgcccct     840tagtgctgca gctaacgcat taagcactcc gcctggggag tacgaccgca ag#gttgaaac     900tcaaaggaat tgacggggac ccgcacaagc ggtggagcat gtggtttaat tc#gaagcaac     960gcgaagaacc ttaccaaatc ttgacatcct ttgacaactc tagagataga gc#cttcccct    1020tcgggggaca aagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt ga#gatgttgg    1080gttaagtccc gcaacgagcg caacccttaa gcttagttgc catcattaag tt#gggcactc    1140taagttgact gccggtgaca aaccggagga aggtggggat gacgtcaaat ca#tcatgccc    1200cttatgattt gggctacaca cgtgctacaa tggacaatac aaagggcagc ga#aaccgcga    1260ggtcaagcaa atcccataaa gttgttctca gttcggattg tagtctgcaa ct#cgactaca    1320tgaagctgga atcgctagta atcgtagatc agcattctac ggtgaatacg tt#cccgggtc    1380ttgtacacac cgcccgtcac accacgagag tttgtaacac ccgaagccgg tg#gagtaacc    1440 ttttaggagc tagccgtcga aggtgggaca aatgattggg gtga   #                   #1484 <210> SEQ ID NO 7 <211> LENGTH: 1464<212> TYPE: DNA <213> ORGANISM: Mycobacterium tuberculosis<400> SEQUENCE: 7ggcggcgtgc ttaacacatg caagtcgaac ggaaaggtct cttcggagat ac#tcgagtgg      60cgaacgggtg agtaacacgt gggtgatctg ccctgcactt cgggataagc ct#gggaaact     120gggtctaata ccggatagga ccacgggatg catgtcttgt ggtggaaagc gc#tttagcgg     180tgtgggatga gcccgcggcc tatcagcttg ttggtggggt gacggcctac ca#aggcgacg     240acgggtagcc ggcctgagag ggtgtccggc cacactggga ctgagatacg gc#ccagactc     300ctacgggagg cagcagtggg gaatattgca caatgggcgc aagcctgatg ca#gcgacgcc     360gcgtggggga tgacggcctt cgggttgtaa acctctttca ccatcgacga ag#gtccgggt     420tctctcggat tgacggtagg tggagaagaa gcaccggcca actacgtgcc ag#cagccgcg     480gtaatacgta gggtgcgagc gttgtccgga attactgggc gtaaagagct cg#taggtggt     540ttgtcgcgtt gttcgtgaaa tctcacggct taactgtgag cgtgcgggcg at#acgggcag     600actagagtac tgcaggggag actggaattc ctggtgtagc ggtggaatgc gc#agatatca     660ggaggaacac cggtggcgaa ggcgggtctc tgggcagtaa ctgacgctga gg#agcgaaag     720cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacgg tg#ggtactag     780gtgtgggttt ccttccttgg gatccgtgcc gtagctaacg cattaagtac cc#cgcctggg     840gagtacggcc gcaaggctaa aactcaaagg aattgacggg ggcccgcaca ag#cggcggag     900catgtggatt aattcgatgc aacgcgaaga accttacctg ggtttgacat gc#acaggacg     960cgtctagaga taggcgttcc cttgtggcct gtgtgcaggt ggtgcatggc tg#tcgtcagc    1020tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgtc tc#atgttgcc    1080agcacgtaat ggtggggact cgtgagagac tgccggggtc aactcggagg aa#ggtgggga    1140tgacgtcaag tcatcatgcc ccttatgtcc agggcttcac acatgctaca at#ggccggta    1200caaagggctg cgatgccgcg aggttaagcg aatccttaaa agccggtctc ag#ttcggatc    1260ggggtctgca actcgacccc gtgaagtcgg agtcgctagt aatcgcagat ca#gcaacgct    1320gcggtgaata cgttcccggg ccttgtacac accgcccgtc acgtcatgaa ag#tcggtaac    1380acccgaagcc agtggcctaa ccctcgggag ggagctgtcg aaggtgggat cg#gcgattgg    1440 gacgaagtcg taacaaggta gccg          #                   #               1464 <210> SEQ ID NO 8<211> LENGTH: 1450 <212> TYPE: DNA <213> ORGANISM: Helicobacter pylori<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1450)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 8tttatggaga gtttgatcct ggctcagagt gaacgctggc ggcgtgccta at#acatgcaa      60gtcgaacgat gaagcttcta gcttgctaga gtgctgatta gtggcgcacg gg#tgagtaac     120gcataggtca tgtgcctctt agtttgggat agccattgga aacgatgatt aa#taccagat     180actcctacgg gggaaagatt tatcgctaag agatcagcct atgtcctatc ag#cttgttgg     240taaggtaatg gcttaccaag gctatgacgg gtatccggcc tgagagggtg aa#cggacaca     300ctggaactga gacacggtcc agactcctac gggaggcagc agtagggaat at#tgctcaat     360gggggaaacc ctgaagcagc aacgccgcgt ggaggatgaa ggttttagga tt#gtaaactc     420cttttgttag agaagataat gacggtatct aacgaataag caccggctaa ct#ccgtgcca     480gcagccgcgg taatacggag ggtgcaagcg ttactcggaa tcactgggcg ta#aagagcgc     540gtaggcggga tagtcagtca ggtgtgaaat cctatggctt aaccatagaa ct#gcatttga     600aactactatt ctagagtgtg ggagaggtag gtggaattct tggtgtaggg gt#aaaatccg     660tagagatcaa gaggaatact cattgcgaag gcgacctgct ggaacattac tg#acgctgat     720tgcgctaaag cgtggggagc aaacaggatt agataccctg gtagtccacg cc#ctaaacga     780tggatgctag ttgttggagg gcttagtctc tccagtaatg cagctaacgc at#taagcatc     840ccgcctgggg agtacggtcg caagattaaa actcaaagga atagacgggg ac#ccgcacaa     900gcggtggagc angtggttta attcgannnn acacgaagaa ccttacctag gc#ttgacatt     960gagagaatcc gctagaaata gtggagtgtc tagcttgcta gaccttgaaa ac#aggtgctg    1020cacggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac ga#gcgcaacc    1080ccntttctta gttgctaaca ggttatgctg agaactctaa ggatactgcc tc#cgtaagga    1140ggaggaaggt ggggacgacg tcaagtcatc atggccctta cgcctagggc ta#cacacgtg    1200ctacaatggg gtgcacaaag agaagcaata ctgtgaagtg gagccaatct tc#aaaacacc    1260tctcagttcg gattgtaggc tgcaactcgc ctgcatgaag ctggaatcgc ta#gtaatcgc    1320aaatcagcca tgttgcggtg aatacgttcc cgggtcttgt actcaccgcc cg#tcacacca    1380tgggagttgt gtttgcctta agtcaggatg ctaaattggc tactgcccac gg#cacacaca    1440 gcgactgggg                 #                  #                   #       1450 <210> SEQ ID NO 9 <211> LENGTH: 1515<212> TYPE: DNA <213> ORGANISM: Streptococcus pneumoniae<400> SEQUENCE: 9atttgatcct ggctcaggac gaacgctggc ggcgtgccta atacatgcaa gt#agaacgct      60gaaggaggag cttgcttctc tggatgagtt gcgaacgggt gagtaacgcg ta#ggtaacct     120gcctggtagc gggggataac tattggaaac gatagctaat accgcataag ag#tggatgtt     180gcatgacatt tgcttaaaag gtgcacttgc atcactacca gatggacctg cg#ttgtatta     240gctagttggt ggggtaacgg ctcaccaagg cgacgataca tagccgacct ga#gagggtga     300tcggccacac tgggactgag acacgkccca gactcctacg ggaggcagca gt#agggaatc     360ttcggcaatg gacggaagtc tgaccgagca acgccgcgtg agtgaagaag gt#tttcggat     420cgtaaagctc tgttgtaaga gaagaacgag tgtgagagtg gaaagttcac ac#tgtgacgg     480tatcttacca gaaagggacg gctaactacg tgccagcagc cgcggtaata cg#taggtccc     540gagcgttgtc cggatttatt gggcgtaaag cgagcgcagg cggttagata ag#tctgaagt     600taaaggctgt ggcttaacca tagtaggctt tggaaactgt ttaacttgag tg#caagaggg     660gagagtggaa ttccatgtgt agcggtgaaa tgcgtagata tatggaggaa ca#ccggtggc     720gaaagcggct ctctggcttg taactgacgc tgaggctcga aagcgtgggg ag#caaacagg     780attagatacc ctggtagtcc acgctgtaaa cgatgagtgc taggtgttag ac#cctttccg     840gggtttagtg ccgtagctaa cgcattaagc actccgcctg gggagtacga cc#gcaaggtt     900gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt tt#aattcgaa     960gcaacgcgaa gaaccttacc aggtcttgac atccctctga ccgctctaga ga#tagagttt    1020tccttcggga cagaggtgac aggtggtgca tggttgtcgt cagctcgtgt cg#tgagatgt    1080tgggttaagt cccgcaacga gcgcaacccc tattgttagt tgccatcatt ca#gttgggca    1140ctctagcgag actgccggta ataaaccgga ggaaggtggg gatgacgtca aa#tcatcatg    1200ccccttatga cctgggctac acacgtgcta caatggctgg tacaacgagt cg#caagccgg    1260tgacggcaag ctaatctctt aaagccagtc tcagttcgga ttgtaggctg ca#actcgcct    1320acatgaagtc ggaatcgcta gtaatcgcgg atcagcacgc cgcggtgaat ac#gttcccgg    1380gccttgtaca caccgcccgt cacaccacga gagtttgtaa cacccgaagt cg#gtgaggta    1440accgtaagga gccagccgcc taaggtggga tagatgattg gggtgaagtc gt#aacaaggt    1500 cagccgtttg ggaga               #                  #                   #   1515 <210> SEQ ID NO 10 <211> LENGTH: 1544<212> TYPE: DNA <213> ORGANISM: Treponema palladium <400> SEQUENCE: 10agagtttgat catggctcag aacgaacgct ggcggcgcgt cttaagcatg ca#agtcgaac      60ggcaagagag gagcttgctt ctctcctaga gtggcggact ggtgaggaac ac#gtgggtaa     120tctaccctta agatggggat agctgctaga aatagcaggt aataccgaat at#actcagtg     180cttcataagg ggtattgagg aaaggaagct acggcttcgc ttgaggatga gc#ttgcgtcc     240cattagctag ttggtgaggt aaaggcccac caaggcgacg atgggtatcc gg#cctgagag     300ggtgatcrga cacattggga ctgagatacg gcccaaactc ctacgggagg ca#gcagctaa     360gaatattccg caatggacgg aagtctgacg gagcgacgcc gcgtggatga ag#aaggctga     420aaagttgtaa aatccttttg ttgatgaaga ataagggtga gagggaatgc tc#atctgatg     480acggtaatcg acgaataagc cccggctaat tacgtgccag cagccgcggt aa#cacgtaag     540gggcgagcgt tgttcggaat tattgggcgt aaagggcatg taggcggtta tg#taagcctg     600atgtgaaatc ctggggctta accccagaat agcattgggt actgtgtaac tt#gaattacg     660gaagggaaac tggaattcca agtgtagggg tggaatctgt agatatttgg aa#gaacaccg     720gtggcgaagg cgggtttctg gccgataatt gacgctgaga tgcgaaagtg tg#gggatcga     780acaggattag ataccctggt agtccacacc gtaaacgatg tacactaggt gt#tggggcaa     840gagcttcagt gccaaagcaa acgcgataag tgtaccgcct ggggagtatg cc#cgcaaggg     900tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg tt#taattcga     960tggtacgcga ggaaccttac ctgggtttga catctagtag aaggtcttag ag#ataaggcc    1020gggtagcaat accctgctag acaggtgctg catggctgtc gtcagctcgt gc#cgtgaggt    1080gttgggttaa gtcccgcaat gagcgcaacc cctactgcca gttactaaca gg#taaagctt    1140gaggactctg gcggaactgc cgatgacaaa tcggaggaag gtggggatga cg#tcaagtca    1200tcatggccct tatgtccagg gctacacacg tgctacaatg gttgctacaa ag#cgaagcaa    1260gaccgtaagg tggagcaagc cgcaaaaaag caatcgtagt tcggattgaa gt#ctgaaact    1320cgacttcatg aagttggaat cgctagtaat cgcgcatcag cacggcgcgg tg#aatacgtt    1380cccgggcctt gtacacaccg cccgtcacac catccgagtt gggggtaccc ga#agtcgctt    1440gtctaacctg caaaggagga cggtgccgaa ggtacgcttg gtaaggaggg tg#aagtcgta    1500 acaaggtagc cgtaccggaa ggtgcggctg gatcacctcc ttaa   #                   #1544 <210> SEQ ID NO 11 <211> LENGTH: 1548<212> TYPE: DNA <213> ORGANISM: Chlamydia trachomatis <400> SEQUENCE: 11ctgagaattt gatcttggtt cagattgaac gctggcggcg tggatgaggc at#gcaagtcg      60aacggagcaa ttgtttcggc aattgtttag tggcggaagg gttagtaatg ca#tagataat     120ttgtccttaa cttgggaata acggttggaa acggccgcta ataccgaatg tg#gcgatatt     180tgggcatccg agtaacgtta aagaagggga tcttaggacc tttcggttaa gg#gagagtct     240atgtgatatc agctagttgg tggggtaaag gcctaccaag gctatgacgt ct#aggcggat     300tgagagattg gccgccaaca ctgggactga gacactgccc agactcctac gg#gaggctgc     360agtcgagaat ctttcgcaat ggacggaagt ctgacgaagc gacgccgcgt gt#gtgatgaa     420ggctctaggg ttgtaaagca ctttcgcttg ggaataagag aagacggtta at#acccgctg     480gatttgagcg taccaggtaa agaagcaccg gctaactccg tgccagcagc tg#cggtaata     540cggagggtgc tagcgttaat cggatttatt ggccgtaaag gccgtgtagg cg#gaaaggta     600agttagttgt caaagatcgg ggctcaaccc cgagtcggca tctaatacta tt#tttctaga     660gggtagatgg agaaaaggga atttcacgtg tagcggtgaa atgcgtagat at#gtggaaga     720acaccagtgg cgaaggcgct tttctaattt atacctgacg ctaaggcgcg aa#agcaaggg     780gagcaaacag gattagatac cctggtagtc cttgccgtaa acgatgcata ct#tgatgtgg     840atggtctcaa ccccatccgt gtcggagcta acgcgttaag tatgccgcct ga#ggagtaca     900ctcgcaaggg tgaaactcaa aagaattgac gggggcccgc acaagcagtg ga#gcatgtgg     960tttaattcga tgcaacgcga aggaccttac ctgggtttga catgtatatg ac#cgcggcag    1020aaatgtcgtt ttccgcaagg acatatacac aggtgctgca tggctgtcgt ca#gctcgtgc    1080cgtgaggtgt tgggttaagt cccgcaacga gcgcaaccct tatcgttagt tg#ccagcact    1140tagggtggga actctaacga gactgcctgg gttaaccagg aggaaggcga gg#atgacgtc    1200aagtcagcat ggcccttatg cccagggcga cacacgtgct acaatggcca gt#acagaagg    1260tagcaagatc gtgagatgga gcaaatcctc aaagctggcc ccagttcgga tt#gtagtctg    1320caactcgact acatgaagtc ggaattgcta gtaatggcgt gtcagccata ac#gccgtgaa    1380tacgttcccg ggccttgtac acaccgcccg tcacatcatg ggagttggtt tt#accttaag    1440tcgttgactc aacccgcaag gagagaggcg cccaaggtga ggctgatgac ta#ggatgaag    1500 tcgtaacaag gtagccctac cggaaggtgg ggctggatca cctccttt  #               1548 <210> SEQ ID NO 12 <211> LENGTH: 1466<212> TYPE: DNA <213> ORGANISM: Bartonella henselae <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1466)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 12tcctggctca ggatgaacgc tggcggcagg cttaacacat gcaagtcgag cg#cactcatt     60tagagtgagc ggcagacggg tgagtaacgc gtgggaatct acccttttct ac#ggaataac    120acagagaaat ttgtgctaat accgtatacg tcctactgga gaaagattta tc#ggagaagg    180atgagcccgc gttggattag ctagttggtg aggtaaaggc tcaccaaggc ga#cgatccat    240agctggtctg agaggatgat cagccacact gggactgaga cacggcccag ac#tcctacgg    300gaggcagcag tggggaatat tggacaatgg gggcaaccct gatccagcca tg#ccgcgtga    360gtgatgaagg ccctagggtt gtaaagctct ttcaccggtg aagataatga cg#gtaaccgg    420agaagaagcc ccggctaact tcgtgccagc agccgcggta atacgaaggg gg#ctagcgtt    480gttcggattt actgggcgta aagcgcatgt aggcggatat ttaagtcaga gg#tgaaatcc    540cagggctcaa ccctggaact gcctttgata ctggatatct tgagtatgga ag#aggtgagt    600ggaattccga gtgtagaggt aaaattcgta gatattcgga ggaacaccag tg#gcgaaggc    660ggctcactgg tccattactg acgctgaggt gcgaaagcgt ggggagcaaa ca#ggattaga    720taccctggta gtccacgccg taaacgatga atgttagccg ttgggtggtt ta#ctgctcag    780tggcgcacgt aacgcattaa acattccgcc tggggagtac ggtcgcaaga tt#aaaactca    840aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aa#gcaacgcg    900cagaacctta ccagcccttg acatcccgat cgcgggaagt ggagacaccc tc#cttcagtt    960cggctggatc ggagacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg ag#atgttggg   1020ttaagtcccg caacgagcgc aaccctcgcc cttagttgcc agcattcagt tg#ggcactct   1080agggggactg ccggtgataa gccgagagga aggtggggat gacgtcaagt cc#tcatggcc   1140cttacgggct gggctacaca cgtgctacaa tggtggtgac agtgggcagc ga#gatcgcaa   1200ggtcgagcta atctccaaaa gccatctcag ttcggattgc actctgcaac tc#gagtgcat   1260gaagttggaa tcgctagtaa tcgtggatca gcatgctacg gtgaatacgt nc#ccgggcct   1320tgtacacacc gcccgtcaca ccatgggagt tggttttacc cgaaggtgct gt#gctaaccg   1380caaggaggca ggtaaccacg gtagggtcag cgactggggt gaagtcgtaa ca#aggtagcc   1440 gtagggaacc tgcggctgga tcacct          #                   #            1466 <210> SEQ ID NO 13<211> LENGTH: 1487 <212> TYPE: DNA <213> ORGANISM: Hemophilis influenza<220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1487)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 13naattgaaga gtttgatcat ggctcagatt gaacgctggc ggcaggctta ac#acatgcaa     60gtcgaacggt agcaggagaa agcttgcttt cttgctgacg agtggcggac gg#gtgagtaa    120tgcttgggaa tctggcttat ggagggggat aacgacggga aactgtcgct aa#taccgcgt    180attatcggaa gatgaaagtg cgggactgag aggccgcatg ccataggatg ag#cccaagtg    240ggattaggta gttggtgggg taaatgccta ccaagcctgc gatctctagc tg#gtctgaga    300ggatgaccag ccacactgga actgagacac ggtccagact cctacgggag gc#agcagtgg    360ggaatattgc gcnatggggg gaaccctgac gcagccatgc cgcgtgaatg aa#gaaggcc     420tcgggttgta aagttctttc ggtattgagg aaggttgatg tgttaatagc ac#atcaaat     480gacgttaaat acagaagaag caccggctaa ctccgtgcca gcagccgcgg ta#atacgag     540ngtgcgagcg ttaatcggaa taactgggcg taaagggcac gcaggcggtt at#ttaatga     600ggtgtgaaag ccccgggctt aacctgggna ttgcatttca gactgggtaa ct#agatact     660ttagggaggg gtagaattcc acgtgtagcg gtgaaatgcg tagagatgtg ga#ggatacc     720gaaggcgaag gcagcccctt gggaatgtac tgacgctcat gtgcgaaagc gt#gggagca     780aacaggatta gataccctgg tagtccacgc tgtaaacgct gtcgatttgg gg#ttggggt     840ttaactctgg cacccgtagc taacgtgata aatcgaccgc ctggggagta cg#ccgcaag     900gttaaaactc aaatgaattg acgggggccn gcacaagcgg tggagcatgt gt#ttaattc     960gatgcaacgc gaagaacctt acctactctt gacatcctaa gaagagctcagagatg#agct    1020 tgtgccttcg ggaacttaga gacaggtgct gcatggctgt cgtcagctcg tg#ttgtgaaa   1080tgttgggtta agtcccgcaa cgagcgcaac ccttatcctt tgttgccagc ga#cttggtcg   1140ggaactcaaa ggagactgcc agtgataaac tggaggaagg tngggatgac gt#caagtcat   1200catggccctt acgagtaggg ctacacacgt gctacaatgg cgtatacaga gg#gaagcgaa   1260gctgcgaggt ggagcgaatc tcataaagta cgtctaagtc cggattggag tc#tgcaactc   1320gactccatga agtcggaatc gctagtaatc gcgaatcaga atgtcgcggt ga#atacgttc   1380ccgggcnttg tacacaccgc ccgtcacacc atgggagtgg gttgtaccag aa#gtagatag   1440 cttaaccttt tggagggcgt ttaccacggt atgattcatg actgggg   #              1487 <210> SEQ ID NO 14 <211> LENGTH: 1487<212> TYPE: DNA <213> ORGANISM: Shigella dysenterae <400> SEQUENCE: 14tggctcagat tgaacgctgg cggcaggcct aacacatgca agtcgaacgg ta#acagaaag     60cagcttgctg tttgctgacg agtggcggac gggtgagtaa tgtctgggaa ac#tgcctgat    120ggagggggat aactactgga aacggtagct aataccgcat aacgtcgcaa ga#ccaaagag    180ggggaccttc gggcctcttg ccatcggatg tgcccagatg ggattagcta gt#aggtgggg    240taacggctca cctaggcgac gatccctagc tggtctgaga ggatgaccag cc#acactgga    300actgagacac ggtccagact cctacgggag gcagcagtgg ggaatattgc ac#aatgggcg    360caagcctgat gcagccatgc cgcgtgtatg aagaaggcct tcgggttgta aa#gtactttc    420agcggggagg aagggagtaa agttaatacc tttgctcatt gacgttaccc gc#agaagaag    480caccggctaa ctccgtgcca gcagccgcgg taatacggag ggtgcaagcg tt#aatcggaa    540ttactgggcg taaagcgcac gcaggcggtt tgttaagtca gatgtgaaat cc#ccgggctc    600aacctgggaa ctgcatctga tactggcaag cttgagtctc gtagaggggg gt#agaattcc    660aggtgtagcg gtgaaatgcg tagagatctg gaggaatacc ggtggcgaag gc#ggccccct    720ggacgaaaac tgacgctcag gtgcgaaagc gtggggagca aacaggatta ga#taccctgg    780tagtccacgc cgtaaacgat gtcgacttgg aggttgtgcc cttgaggcgt gg#cttccgga    840gctaacgcgt taagtcgacc gcctggggag tacggccgca aggttaaaac tc#aaatgaat    900tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgatgcaac gc#gaagaacc    960ttacctggtc ttgacatcca cagaaccttg tagagatacg agggtgcctt cg#ggaactgt   1020gagacaggtg ctgcatggct gtcgtcagct cgtgttgtga aatgttgggt ta#agtcccgc   1080aacgagcgca acccttatcc tttgttgcca gcggtccggc cgggaactca aa#ggagactg   1140ccagtgataa actggaggaa ggtggggatg acgtcaagtc atcatggccc tt#acgaccag   1200ggctacacac gtgctacaat ggcgcataca aagagaagcg acctcgcgag ag#caagcgga   1260cctcataaag tgcgtcgtag tccggattgg agtctgcaac tcgactccat ga#agtcggaa   1320tcgctagtaa tcgtggatca gaatgtcacg gtgaatacgt tcccgggcct tg#tacacacc   1380gcccgtcaca ccatgggagt gggttgcaaa agaagtaggt agcttaacct tc#gggagggc   1440 gcttaccact ttgtgattca tgactggggt gaagtcgtaa caaggta   #              1487 <210> SEQ ID NO 15 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 15ggactacgac gcactttatg ag            #                  #                 22 <210> SEQ ID NO 16 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 16ggtccgcttg ctctcgcgag g            #                  #                   #21 <210> SEQ ID NO 17 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 17gcaaaggtat taactttact c            #                  #                   #21 <210> SEQ ID NO 18 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 18gctgcggtta ttaaccacaa c            #                  #                   #21 <210> SEQ ID NO 19 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 19gcactttatg aggtccgctt g            #                  #                   #21 <210> SEQ ID NO 20 <220> FEATURE:<223> OTHER INFORMATION: No sequence is present <211> LENGTH:<212> TYPE: <213> ORGANISM: <400> SEQUENCE: 20 000 <210> SEQ ID NO 21<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 21 tgctgcctcc cgtaggagtc t           #                   #                   #21 <210> SEQ ID NO 22<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 22 attaccgcgg ctgctggcac g           #                   #                   #21 <210> SEQ ID NO 23<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 23 accagggtat ctaatcctgt t           #                   #                   #21 <210> SEQ ID NO 24<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 24 cacatgctcc accgcttgtg c           #                   #                   #21 <210> SEQ ID NO 25<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 25 ttgcgggact taacccaaca t           #                   #                   #21 <210> SEQ ID NO 26<220> FEATURE: <223> OTHER INFORMATION: No sequence is present<211> LENGTH: <212> TYPE: <213> ORGANISM: <400> SEQUENCE: 26 000<210> SEQ ID NO 27 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 27cgcggctgct ggcacgtagt t            #                  #                   #21 <210> SEQ ID NO 28 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 28acttaaccca acatctcacg a            #                  #                   #21 <210> SEQ ID NO 29 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 29tttacgccca gtaattccga             #                  #                   # 20 <210> SEQ ID NO 30 <211> LENGTH: 22<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 30actcccatgg tgtgacgggc gg            #                  #                 22 <210> SEQ ID NO 31 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 31aatctgagcc atgatcaaac t            #                  #                   #21 <210> SEQ ID NO 32 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 32ccctctttgt gcttgcgacg t            #                  #                   #21 <210> SEQ ID NO 33 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 33acccccctct acgagactca a            #                  #                   #21 <210> SEQ ID NO 34 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 34ccacgcctca agggcacaac c            #                  #                   #21 <210> SEQ ID NO 35 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 35tctcatctct gaaaacttcc g            #                  #                   #21 <210> SEQ ID NO 36 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 36catgatcaaa ctcttcaatt t            #                  #                   #21 <210> SEQ ID NO 37 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 37ccctctttgg tcttgcgacg t            #                  #                   #21 <210> SEQ ID NO 38 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 38tacccccctc tacgagactc a            #                  #                   #21 <210> SEQ ID NO 39 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 39gccacgcctc aagggcacaa c            #                  #                   #21 <210> SEQ ID NO 40 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 40cagagagcaa gccctcttca t            #                  #                   #21 <210> SEQ ID NO 41 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 41cctgctttct cccgtaggac g            #                  #                   #21 <210> SEQ ID NO 42 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 42caccaccctc tgccatactc t            #                  #                   #21 <210> SEQ ID NO 43 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 43ctaagatctc aaggatccca a            #                  #                   #21 <210> SEQ ID NO 44 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 44ggcctgccgc cagcgttcaa t            #                  #                   #21 <210> SEQ ID NO 45 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 45ccctctttgg tccgtaaaca t            #                  #                   #21 <210> SEQ ID NO 46 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 46ccccctctac aagactctag c            #                  #                   #21 <210> SEQ ID NO 47 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 47acgactytag gtcacaacct c            #                  #                   #21 <210> SEQ ID NO 48 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 48aggatcaaac tcttatgttc a            #                  #                   #21 <210> SEQ ID NO 49 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 49cctgctttcc ctctcaagac g            #                  #                   #21 <210> SEQ ID NO 50 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 50cacctccctc tgacacactc g            #                  #                   #21 <210> SEQ ID NO 51 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 51ccaagcaatc aagttgccca a            #                  #                   #21 <210> SEQ ID NO 52 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 52ccagcgttca tcctgagcca g            #                  #                   #21 <210> SEQ ID NO 53 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 53gaaccatgcg gttcaaaata t            #                  #                   #21 <210> SEQ ID NO 54 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 54ctttcctctt ctgcactcaa g            #                  #                   #21 <210> SEQ ID NO 55 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 55ggggcggaaa ccccctaaca c            #                  #                   #21 <210> SEQ ID NO 56 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 56gcatgtgtta agcacgccgc c            #                  #                   #21 <210> SEQ ID NO 57 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 57aagacatgca tcccgtggtc c            #                  #                   #21 <210> SEQ ID NO 58 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 58cagtctcccc tgcagtactc t            #                  #                   #21 <210> SEQ ID NO 59 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 59gatcccaagg aaggaaaccc a            #                  #                   #21 <210> SEQ ID NO 60 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 60caggatcaaa ctctccataa a            #                  #                   #21 <210> SEQ ID NO 61 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 61aaatctttcc cccgtaggag t            #                  #                   #21 <210> SEQ ID NO 62 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 62cacctacctc tcccacactc t            #                  #                   #21 <210> SEQ ID NO 63 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 63tggagagact aagccctcca a            #                  #                   #21 <210> SEQ ID NO 64 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 64cgtcctgagc caggatcaaa t            #                  #                   #21 <210> SEQ ID NO 65 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 65atgtcatgca acatccactc t            #                  #                   #21 <210> SEQ ID NO 66 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 66actctcccct cttgcactca a            #                  #                   #21 <210> SEQ ID NO 67 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 67aaaccccgga aagggtctaa c            #                  #                   #21 <210> SEQ ID NO 68 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 68tctgagccat gatcaaactc t            #                  #                   #21 <210> SEQ ID NO 69 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 69accccttatg aagcactgag t            #                  #                   #21 <210> SEQ ID NO 70 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 70agtttccctt ccgtaattca a            #                  #                   #21 <210> SEQ ID NO 71 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 71cactgaagct cttgccccaa c            #                  #                   #21 <210> SEQ ID NO 72 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 72gaaccaagat caaattctca g            #                  #                   #21 <210> SEQ ID NO 73 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 73gttactcgga tgcccaaata t            #                  #                   #21 <210> SEQ ID NO 74 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 74ccttttctcc atctaccctc t            #                  #                   #21 <210> SEQ ID NO 75 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 75ggatggggtt gagaccatcc a            #                  #                   #21 <210> SEQ ID NO 76 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 76agcgttcatc ctgagccagg a            #                  #                   #21 <210> SEQ ID NO 77 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 77aaatctttct ccagtaggac g            #                  #                   #21 <210> SEQ ID NO 78 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 78cactcacctc ttccatactc a            #                  #                   #21 <210> SEQ ID NO 79 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 79actgagcagt aaaccaccca a            #                  #                   #21 <210> SEQ ID NO 80 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <221> NAME/KEY: misc_feature<222> LOCATION: (1)...(21) <223> OTHER INFORMATION: n = A,T,C or G<400> SEQUENCE: 80 catgatcaaa ctcttcaatt n           #                   #                   # 21 <210> SEQ ID NO 81<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 81 cactttcatc ttccgataat a           #                   #                   # 21 <210> SEQ ID NO 82<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 82 cctccctaaa gtactctagt t           #                   #                   # 21 <210> SEQ ID NO 83<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 83 cagagttaaa ccccaacccc c           #                   #                   # 21 <210> SEQ ID NO 84<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 84 gccagcgttc aatctgagcc a           #                   #                   # 21 <210> SEQ ID NO 85<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 85 ccctctttgg tcttgcgacg t           #                   #                   # 21 <210> SEQ ID NO 86<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 86 tacccccctc tacgagactc a           #                   #                   # 21 <210> SEQ ID NO 87<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 87 gccacgcctc aagggcacaa c           #                   #                   # 21 <210> SEQ ID NO 88<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 88 cctcgtatct ctacaaggtt c           #                   #                   # 21 <210> SEQ ID NO 89<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 89 ccccatcatt atgagtgatg tgc           #                   #                 23 <210> SEQ ID NO 90<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense oligomer<400> SEQUENCE: 90 tcattatgag gtgacccca              #                  #                   #  19 <210> SEQ ID NO 91 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 91gatgaacagt tactctcatc             #                  #                   #  20 <210> SEQ ID NO 92 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 92actgagagaa gctttaagag             #                  #                   #  20 <210> SEQ ID NO 93 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 93atgtgcacag ttacttacac             #                  #                   #  20 <210> SEQ ID NO 94 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 94ctgagaacaa ctttatggga             #                  #                   #  20 <210> SEQ ID NO 95 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 95ttattctgtt ggtaacgtca             #                  #                   #  20 <210> SEQ ID NO 96 <211> LENGTH: 19<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 96cgagttgcag actgcgatc              #                  #                   #  19 <210> SEQ ID NO 97 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 97atctgagcca tgatcaaact             #                  #                   #  20 <210> SEQ ID NO 98 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 98tgtctcagtt ccagtgttgc             #                  #                   #  20 <210> SEQ ID NO 99 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 99gtcttcgtcc agggggccgc             #                  #                   #  20 <210> SEQ ID NO 100 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 100cacctgtctc acggttcccg             #                  #                   #  20 <210> SEQ ID NO 101 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 101cgccctcccg aagttaagct             #                  #                   #  20 <210> SEQ ID NO 102 <211> LENGTH: 19<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 102ggcacgccgc cagcgttcg              #                  #                   #  19 <210> SEQ ID NO 103 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 103tgtctcagtc ccaatgtggc             #                  #                   #  20 <210> SEQ ID NO 104 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 104gttacagacc agagagccgc             #                  #                   #  20 <210> SEQ ID NO 105 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 105cacctgtcac tttgcccccg             #                  #                   #  20 <210> SEQ ID NO 106 <211> LENGTH: 19<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 106ggcggctggc tccaaaagg              #                  #                   #  19 <210> SEQ ID NO 107 <211> LENGTH: 19<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 107cacccgttcg ccactcctc              #                  #                   #  19 <210> SEQ ID NO 108 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 108tcaattcctt tgagtttcaa             #                  #                   #  20 <210> SEQ ID NO 109 <211> LENGTH: 29<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 109gcaatccgaa ctgagagaag ctttaagag          #                  #             29 <210> SEQ ID NO 110 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 110ccgaactgag agaagcttta agag           #                  #                 24 <210> SEQ ID NO 111 <211> LENGTH: 17<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 111gagagaagct ttaagag              #                   #                  #    17 <210> SEQ ID NO 112 <211> LENGTH: 15 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 112gagaagcttt aagag               #                   #                  #     15 <210> SEQ ID NO 113 <211> LENGTH: 12 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 113aagctttaag ag               #                   #                  #        12 <210> SEQ ID NO 114 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 114gactaccagg gtatctaatc             #                  #                   #  20 <210> SEQ ID NO 115 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 115cagcgacacc cgaaagcgcc             #                  #                   #  20 <210> SEQ ID NO 116 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 116gtgccaaggc atccaccgtg             #                  #                   #  20 <210> SEQ ID NO 117 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 117catactcaaa cgccctattc             #                  #                   #  20 <210> SEQ ID NO 118 <211> LENGTH: 19<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 118ccttagcctc ctgcgtccc              #                  #                   #  19 <210> SEQ ID NO 119 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 119ggggtctttc cgtcctgtcg             #                  #                   #  20 <210> SEQ ID NO 120 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 120cgatcgatta gtatcagtcc             #                  #                   #  20 <210> SEQ ID NO 121 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 121tgagagaagc tttaagag              #                   #                  #   18 <210> SEQ ID NO 122 <211> LENGTH: 19 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 122ctgagagaag ctttaagag              #                  #                   #  19 <210> SEQ ID NO 123 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 123gcgacacccg aaagcgcc              #                   #                  #   18 <210> SEQ ID NO 124 <211> LENGTH: 18 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 124tacagaccag agagccgc              #                   #                  #   18 <210> SEQ ID NO 125 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 125cgacacccga aagcgcc              #                   #                  #    17 <210> SEQ ID NO 126 <211> LENGTH: 19 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 126agcgacaccc gaaagcgcc              #                  #                   #  19 <210> SEQ ID NO 127 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 127cgacacccga aagcgcct              #                   #                  #   18 <210> SEQ ID NO 128 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 128mgamammmga aagmgmm              #                   #                  #    17 <210> SEQ ID NO 129 <211> LENGTH: 18 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 129tamagammag agagmmgm              #                   #                  #   18 <210> SEQ ID NO 130 <211> LENGTH: 16 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 130mmmmammttm mtmmgg              #                   #                  #     16 <210> SEQ ID NO 131 <211> LENGTH: 19 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 131caccgcggcg tgctgatcc              #                  #                   #  19 <210> SEQ ID NO 132 <211> LENGTH: 16<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 132ccccaccttc ctccgg              #                   #                  #     16 <210> SEQ ID NO 133 <211> LENGTH: 18 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 133ccgcttgtgc gggccccc              #                   #                  #   18 <210> SEQ ID NO 134 <211> LENGTH: 18 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 134caccgcggcg tgctgatc              #                   #                  #   18 <210> SEQ ID NO 135 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 135caccgcggcg tgctgat              #                   #                  #    17 <210> SEQ ID NO 136 <211> LENGTH: 18 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 136accgcggcgt gctgatcc              #                   #                  #   18 <210> SEQ ID NO 137 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 137ccgcggcgtg ctgatcc              #                   #                  #    17 <210> SEQ ID NO 138 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 138accgcggcgt gctgatc              #                   #                  #    17 <210> SEQ ID NO 139 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense oligomer <400> SEQUENCE: 139acgttgaggg gcatcgtcgc             #                  #                   # 20

It is claimed:
 1. A method of inhibiting growth of bacteria selectedfrom the group consisting of Escherichia coli, Staphylococcus aureus,Pseudomonas aeruginosa, Enterococcus faecium, and Salmonellatyphimurium, comprising contacting said bacteria in vitro with aneffective amount of an antisense morpholino oligomer containing from 10to 40 nucleotide subunits, each of said subunits comprising a morpholinoring supporting a base-pairing moiety effective to bind by Watson-Crickbase pairing to a respective nucleotide base, said base-pairing moietiesincluding a targeting nucleic acid sequence at least 10 nucleotides inlength which is complementary to a 16S or 23S rRNA nucleic acid sequenceof said bacteria, wherein adjacent subunits are joined by unchargedphosphorodiamidate linkages.
 2. The method of claim 1, wherein saidoligomer is able to hybridize with the bacterial sequence at a T_(m)substantially greater than 37° C.
 3. The method of claim 1, wherein eachlinkage is a phosphorodiamidate linkage in accordance with the structurebelow, where X═NR₂, R is hydrogen or methyl, Y₁═O, Z═O, and P, is apurine or pynmidine base pairing moiety effective to bind, by basespecific hydrogen bonding, to a base in a polynucleotide,


4. The method of claim 1, where the antisense oligomer is 25 or fewerbases in length.
 5. The method of claim 1, wherein the region ofcomplementarity with the target RNA sequence has a length of 13 to 20bases.
 6. The method of claim 1, wherein the targeting sequence isselected from the group consisting of SEQ ID NOs: 91-92, 102-115, and121-138.